Sirtuin modulating compounds

ABSTRACT

Provided herein are novel sirtuin-modulating compounds and methods of use thereof. The sirtuin-modulating compounds may be used for increasing the lifespan of a cell, and treating and/or preventing a wide variety of diseases and disorders including, for example, diseases or disorders related to aging or stress, diabetes, obesity, neurodegenerative diseases, cardiovascular disease, blood clotting disorders, inflammation, cancer, and/or flushing as well as diseases or disorders that would benefit from increased mitochondrial activity. Also provided are compositions comprising a sirtuin-modulating compound in combination with another therapeutic agent.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/936,636, filed Jun. 20, 2007, the contents of which are incorporatedby reference in their entirety.

BACKGROUND

The Silent Information Regulator (SIR) family of genes represents ahighly conserved group of genes present in the genomes of organismsranging from archaebacteria to a variety of eukaryotes (Frye, 2000). Theencoded SIR proteins are involved in diverse processes from regulationof gene silencing to DNA repair. The proteins encoded by members of theSIR gene family show high sequence conservation in a 250 amino acid coredomain. A well-characterized gene in this family is S. cerevisiae SIR2,which is involved in silencing HM loci that contain informationspecifying yeast mating type, telomere position effects and cell aging(Guarente, 1999; Kaeberlein et al., 1999; Shore, 2000). The yeast Sir2protein belongs to a family of histone deacetylases (reviewed inGuarente, 2000; Shore, 2000). The Sir2 homolog, CobB, in Salmonellatyphimurium, functions as an NAD (nicotinamide adeninedinucleotide)-dependent ADP-ribosyl transferase (Tsang andEscalante-Semerena, 1998).

The Sir2 protein is a class III deacetylase which uses NAD as acosubstrate (Imai et al., 2000; Moazed, 2001; Smith et al., 2000; Tanneret al., 2000; Tanny and Moazed, 2001). Unlike other deacetylases, manyof which are involved in gene silencing, Sir2 is insensitive to class Iand II histone deacetylase inhibitors like trichostatin A (TSA) (Imai etal., 2000; Landry et al., 2000a; Smith et al., 2000).

Deacetylation of acetyl-lysine by Sir2 is tightly coupled to NADhydrolysis, producing nicotinamide and a novel acetyl-ADP ribosecompound (Tanner et al., 2000; Landry et al., 2000b; Tanny and Moazed,2001). The NAD-dependent deacetylase activity of Sir2 is essential forits functions which can connect its biological role with cellularmetabolism in yeast (Guarente, 2000; Imai et al., 2000; Lin et al.,2000; Smith et al., 2000). Mammalian Sir2 homologs have NAD-dependenthistone deacetylase activity (Imai et al., 2000; Smith et al., 2000).Most information about Sir2 mediated functions comes from the studies inyeast (Gartenberg, 2000; Gottschling, 2000).

Biochemical studies have shown that Sir2 can readily deacetylate theamino-terminal tails of histones H3 and H4, resulting in the formationof 1-O-acetyl-ADP-ribose and nicotinamide. Strains with additionalcopies of SIR2 display increased rDNA silencing and a 30% longer lifespan. It has recently been shown that additional copies of the C.elegans SIR2 homolog, sir-2.1, and the D. melanogaster dSir2 genegreatly extend life span in those organisms. This implies that theSIR2-dependent regulatory pathway for aging arose early in evolution andhas been well conserved. Today, Sir2 genes are believed to have evolvedto enhance an organism's health and stress resistance to increase itschance of surviving adversity.

SIRT3 is a homolog of SIRT1 that is conserved in prokaryotes andeukaryotes (P. Onyango et al., Proc. Natl. Acad. Sci. USA 99:13653-13658 (2002)). The SIRT3 protein is targeted to the mitochondrialcristae by a unique domain located at the N-terminus. SIRT3 hasNAD+-dependent protein deacetylase activity and is ubiquitouslyexpressed, particularly in metabolically active tissues. Upon transferto the mitochondria, SIRT3 is believed to be cleaved into a smaller,active form by a mitochondrial matrix processing peptidase (MPP) (B.Schwer et al., J. Cell Biol. 158: 647-657 (2002)).

Caloric restriction has been known for over 70 years to improve thehealth and extend the lifespan of mammals (Masoro, 2000). Yeast lifespan, like that of metazoans, is also extended by interventions thatresemble caloric restriction, such as low glucose. The discovery thatboth yeast and flies lacking the SIR2 gene do not live longer whencalorically restricted provides evidence that SIR2 genes mediate thebeneficial health effects of this diet (Anderson et al., 2003; Helfandand Rogina, 2004). Moreover, mutations that reduce the activity of theyeast glucose-responsive cAMP (adenosine 3′,5′-monophosphate)-dependent(PKA) pathway extend life span in wild type cells but not in mutant sir2strains, demonstrating that SIR2 is likely to be a key downstreamcomponent of the caloric restriction pathway (Lin et al., 2001).

SUMMARY

Provided herein are novel sirtuin-modulating compounds and methods ofuse thereof.

In one aspect, the invention provides sirtuin-modulating compounds ofStructural Formulas (I)-(VI) as are described in detail below.

In another aspect, the invention provides methods for usingsirtuin-modulating compounds, or compostions comprisingsirtuin-modulating compounds. In certain embodiments, sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteinmay be used for a variety of therapeutic applications including, forexample, increasing the lifespan of a cell, and treating and/orpreventing a wide variety of diseases and disorders including, forexample, diseases or disorders related to aging or stress, diabetes,obesity, neurodegenerative diseases, chemotherapeutic inducedneuropathy, neuropathy associated with an ischemic event, oculardiseases and/or disorders, cardiovascular disease, blood clottingdisorders, inflammation, and/or flushing, etc. Sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteinmay also be used for treating a disease or disorder in a subject thatwould benefit from increased mitochondrial activity, for enhancingmuscle performance, for increasing muscle ATP levels, or for treating orpreventing muscle tissue damage associated with hypoxia or ischemia. Inother embodiments, sirtuin-modulating compounds that decrease the leveland/or activity of a sirtuin protein may be used for a variety oftherapeutic applications including, for example, increasing cellularsensitivity to stress, increasing apoptosis, treatment of cancer,stimulation of appetite, and/or stimulation of weight gain, etc. Asdescribed further below, the methods comprise administering to a subjectin need thereof a pharmaceutically effective amount of asirtuin-modulating compound.

In certain aspects, the sirtuin-modulating compounds may be administeredalone or in combination with other compounds, including othersirtuin-modulating compounds, or other therapeutic agents.

DETAILED DESCRIPTION 1. Definitions

As used herein, the following terms and phrases shall have the meaningsset forth below. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood to one ofordinary skill in the art.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule (such as a nucleicacid, an antibody, a protein or portion thereof, e.g., a peptide), or anextract made from biological materials such as bacteria, plants, fungi,or animal (particularly mammalian) cells or tissues. The activity ofsuch agents may render it suitable as a “therapeutic agent” which is abiologically, physiologically, or pharmacologically active substance (orsubstances) that acts locally or systemically in a subject.

The term “bioavailable” when referring to a compound is art-recognizedand refers to a form of a compound that allows for it, or a portion ofthe amount of compound administered, to be absorbed by, incorporated to,or otherwise physiologically available to a subject or patient to whomit is administered.

“Biologically active portion of a sirtuin” refers to a portion of asirtuin protein having a biological activity, such as the ability todeacetylate. Biologically active portions of a sirtuin may comprise thecore domain of sirtuins. Biologically active portions of SIRT1 havingGenBank Accession No. NP_(—)036370 that encompass the NAD+ bindingdomain and the substrate binding domain, for example, may includewithout limitation, amino acids 62-293 of GenBank Accession No.NP_(—)036370, which are encoded by nucleotides 237 to 932 of GenBankAccession No. NM_(—)012238. Therefore, this region is sometimes referredto as the core domain. Other biologically active portions of SIRT1, alsosometimes referred to as core domains, include about amino acids 261 to447 of GenBank Accession No. NP_(—)036370, which are encoded bynucleotides 834 to 1394 of GenBank Accession No. NM_(—)012238; aboutamino acids 242 to 493 of GenBank Accession No. NP_(—)036370, which areencoded by nucleotides 777 to 1532 of GenBank Accession No.NM_(—)012238; or about amino acids 254 to 495 of GenBank Accession No.NP_(—)036370, which are encoded by nucleotides 813 to 1538 of GenBankAccession No. NM_(—)012238.

The term “companion animals” refers to cats and dogs. As used herein,the term “dog(s)” denotes any member of the species Canis familiaris, ofwhich there are a large number of different breeds. The term “cat(s)”refers to a feline animal including domestic cats and other members ofthe family Felidae, genus Felis.

The terms “comprise” and “comprising” are used in the inclusive, opensense, meaning that additional elements may be included.

“Diabetes” refers to high blood sugar or ketoacidosis, as well aschronic, general metabolic abnormalities arising from a prolonged highblood sugar status or a decrease in glucose tolerance. “Diabetes”encompasses both the type I and type II (Non Insulin Dependent DiabetesMellitus or NIDDM) forms of the disease. The risk factors for diabetesinclude the following factors: waistline of more than 40 inches for menor 35 inches for women, blood pressure of 130/85 mmHg or higher,triglycerides above 150 mg/dl, fasting blood glucose greater than 100mg/dl or high-density lipoprotein of less than 40 mg/dl in men or 50mg/dl in women.

A “direct activator” of a sirtuin is a molecule that activates a sirtuinby binding to it. A “direct inhibitor” of a sirtuin is a moleculeinhibits a sirtuin by binding to it.

The term “ED₅₀” is art-recognized. In certain embodiments, ED₅₀ meansthe dose of a drug which produces 50% of its maximum response or effect,or alternatively, the dose which produces a pre-determined response in50% of test subjects or preparations. The term “LD₅₀” is art-recognized.In certain embodiments, LD₅₀ means the dose of a drug which is lethal in50% of test subjects. The term “therapeutic index” is an art-recognizedterm which refers to the therapeutic index of a drug, defined asLD₅₀/ED₅₀.

The term “hyperinsulinemia” refers to a state in an individual in whichthe level of insulin in the blood is higher than normal.

The term “including” is used to mean “including but not limited to”.“Including” and “including but not limited to” are used interchangeably.

The term “insulin resistance” refers to a state in which a normal amountof insulin produces a subnormal biologic response relative to thebiological response in a subject that does not have insulin resistance.

An “insulin resistance disorder,” as discussed herein, refers to anydisease or condition that is caused by or contributed to by insulinresistance. Examples include: diabetes, obesity, metabolic syndrome,insulin-resistance syndromes, syndrome X, insulin resistance, high bloodpressure, hypertension, high blood cholesterol, dyslipidemia,hyperlipidemia, dyslipidemia, atherosclerotic disease including stroke,coronary artery disease or myocardial infarction, hyperglycemia,hyperinsulinemia and/or hyperproinsulinemia, impaired glucose tolerance,delayed insulin release, diabetic complications, including coronaryheart disease, angina pectoris, congestive heart failure, stroke,cognitive functions in dementia, retinopathy, peripheral neuropathy,nephropathy, glomerulonephritis, glomerulosclerosis, nephrotic syndrome,hypertensive nephrosclerosis some types of cancer (such as endometrial,breast, prostate, and colon), complications of pregnancy, poor femalereproductive health (such as menstrual irregularities, infertility,irregular ovulation, polycystic ovarian syndrome (PCOS)), lipodystrophy,cholesterol related disorders, such as gallstones, cholescystitis andcholelithiasis, gout, obstructive sleep apnea and respiratory problems,osteoarthritis, and prevention and treatment of bone loss, e.g.osteoporosis.

The term “livestock animals” refers to domesticated quadrupeds, whichincludes those being raised for meat and various byproducts, e.g., abovine animal including cattle and other members of the genus Bos, aporcine animal including domestic swine and other members of the genusSus, an ovine animal including sheep and other members of the genusOvis, domestic goats and other members of the genus Capra; domesticatedquadrupeds being raised for specialized tasks such as use as a beast ofburden, e.g., an equine animal including domestic horses and othermembers of the family Equidae, genus Equus.

The term “mammal” is known in the art, and exemplary mammals includehumans, primates, livestock animals (including bovines, porcines, etc.),companion animals (e.g., canines, felines, etc.) and rodents (e.g., miceand rats).

“Obese” individuals or individuals suffering from obesity are generallyindividuals having a body mass index (BMI) of at least 25 or greater.Obesity may or may not be associated with insulin resistance.

The terms “parenteral administration” and “administered parenterally”are art-recognized and refer to modes of administration other thanenteral and topical administration, usually by injection, and includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intra-articulare, subcapsular, subarachnoid, intraspinal, andintrasternal injection and infusion.

A “patient”, “subject”, “individual” or “host” refers to either a humanor a non-human animal.

The term “pharmaceutically acceptable carrier” is art-recognized andrefers to a pharmaceutically-acceptable material, composition orvehicle, such as a liquid or solid filler, diluent, excipient, solventor encapsulating material, involved in carrying or transporting anysubject composition or component thereof. Each carrier must be“acceptable” in the sense of being compatible with the subjectcomposition and its components and not injurious to the patient. Someexamples of materials which may serve as pharmaceutically acceptablecarriers include: (1) sugars, such as lactose, glucose and sucrose; (2)starches, such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)talc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; and (21) other non-toxic compatiblesubstances employed in pharmaceutical formulations.

The term “prophylactic” or “therapeutic” treatment is art-recognized andrefers to administration of a drug to a host. If it is administeredprior to clinical manifestation of the unwanted condition (e.g., diseaseor other unwanted state of the host animal) then the treatment isprophylactic, i.e., it protects the host against developing the unwantedcondition, whereas if administered after manifestation of the unwantedcondition, the treatment is therapeutic (i.e., it is intended todiminish, ameliorate or maintain the existing unwanted condition or sideeffects therefrom).

The term “pyrogen-free”, with reference to a composition, refers to acomposition that does not contain a pyrogen in an amount that would leadto an adverse effect (e.g., irritation, fever, inflammation, diarrhea,respiratory distress, endotoxic shock, etc.) in a subject to which thecomposition has been administered. For example, the term is meant toencompass compositions that are free of, or substantially free of, anendotoxin such as, for example, a lipopolysaccharide (LPS).

“Replicative lifespan” of a cell refers to the number of daughter cellsproduced by an individual “mother cell.” “Chronological aging” or“chronological lifespan,” on the other hand, refers to the length oftime a population of non-dividing cells remains viable when deprived ofnutrients. “Increasing the lifespan of a cell” or “extending thelifespan of a cell,” as applied to cells or organisms, refers toincreasing the number of daughter cells produced by one cell; increasingthe ability of cells or organisms to cope with stresses and combatdamage, e.g., to DNA, proteins; and/or increasing the ability of cellsor organisms to survive and exist in a living state for longer under aparticular condition, e.g., stress (for example, heatshock, osmoticstress, high energy radiation, chemically-induced stress, DNA damage,inadequate salt level, inadequate nitrogen level, or inadequate nutrientlevel). Lifespan can be increased by at least about 20%, 30%, 40%, 50%,60% or between 20% and 70%, 30% and 60%, 40% and 60% or more usingmethods described herein.

“Sirtuin-activating compound” refers to a compound that increases thelevel of a sirtuin protein and/or increases at least one activity of asirtuin protein. In an exemplary embodiment, a sirtuin-activatingcompound may increase at least one biological activity of a sirtuinprotein by at least about 10%, 25%, 50%, 75%, 100%, or more. Exemplarybiological activities of sirtuin proteins include deacetylation, e.g.,of histones and p53; extending lifespan; increasing genomic stability;silencing transcription; and controlling the segregation of oxidizedproteins between mother and daughter cells.

“Sirtuin-inhibiting compound” refers to a compound that decreases thelevel of a sirtuin protein and/or decreases at least one activity of asirtuin protein. In an exemplary embodiment, a sirtuin-inhibitingcompound may decrease at least one biological activity of a sirtuinprotein by at least about 10%, 25%, 50%, 75%, 100%, or more. Exemplarybiological activities of sirtuin proteins include deacetylation, e.g.,of histones and p53; extending lifespan; increasing genomic stability;silencing transcription; and controlling the segregation of oxidizedproteins between mother and daughter cells.

“Sirtuin-modulating compound” refers to a compound of StructuralFormulas (I)-(VI) as described herein. In exemplary embodiments, asirtuin-modulating compound may either up regulate (e.g., activate orstimulate), down regulate (e.g., inhibit or suppress) or otherwisechange a functional property or biological activity of a sirtuinprotein. Sirtuin-modulating compounds may act to modulate a sirtuinprotein either directly or indirectly. In certain embodiments, asirtuin-modulating compound may be a sirtuin-activating compound or asirtuin-inhibiting compound.

“Sirtuin protein” refers to a member of the sirtuin deacetylase proteinfamily, or preferably to the sir2 family, which include yeast Sir2(GenBank Accession No. P53685), C. elegans Sir-2.1 (GenBank AccessionNo. NP_(—)501912), and human SIRT1 (GenBank Accession No. NM_(—)012238and NP_(—)036370 (or AF083106)) and SIRT2 (GenBank Accession No.NM_(—)012237, NM_(—)030593, NP 036369, NP_(—)085096, and AF083107)proteins. Other family members include the four additional yeastSir2-like genes termed “HST genes” (homologues of Sir two) HST1, HST2,HST3 and HST4, and the five other human homologues hSIRT3, hSIRT4,hSIRT5, hSIRT6 and hSIRT7 (Brachmann et al. (1995) Genes Dev. 9:2888 andFrye et al. (1999) BBRC 260:273). Preferred sirtuins are those thatshare more similarities with SIRT1, i.e., hSIRT1, and/or Sir2 than withSIRT2, such as those members having at least part of the N-terminalsequence present in SIRT1 and absent in SIRT2 such as SIRT3 has.

“SIRT1 protein” refers to a member of the sir2 family of sirtuindeacetylases. In one embodiment, a SIRT1 protein includes yeast Sir2(GenBank Accession No. P53685), C. elegans Sir-2.1 (GenBank AccessionNo. NP_(—)501912), human SIRT1 (GenBank Accession No. NM_(—)012238 orNP_(—)036370 (or AF083106)), and human SIRT2 (GenBank Accession No.NM_(—)012237, NM_(—)030593, NP_(—)036369, NP_(—)085096, or AF083107)proteins, and equivalents and fragments thereof. In another embodiment,a SIRT1 protein includes a polypeptide comprising a sequence consistingof, or consisting essentially of, the amino acid sequence set forth inGenBank Accession Nos. NP_(—)036370, NP_(—)501912, NP_(—)085096,NP_(—)036369, or P53685. SIRT1 proteins include polypeptides comprisingall or a portion of the amino acid sequence set forth in GenBankAccession Nos. NP_(—)036370, NP_(—)501912, NP_(—)085096, NP_(—)036369,or P53685; the amino acid sequence set forth in GenBank Accession Nos.NP_(—)036370, NP_(—)501912, NP_(—)085096, NP_(—)036369, or P53685 with 1to about 2, 3, 5, 7, 10, 15, 20, 30, 50, 75 or more conservative aminoacid substitutions; an amino acid sequence that is at least 60%, 70%,80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to GenBank Accession Nos.NP_(—)036370, NP_(—)501912, NP_(—)085096, NP_(—)036369, or P53685, andfunctional fragments thereof. Polypeptides of the invention also includehomologs (e.g., orthologs and paralogs), variants, or fragments, ofGenBank Accession Nos. NP_(—)036370, NP_(—)501912, NP 085096,NP_(—)036369, or P53685.

“SIRT3 protein” refers to a member of the sirtuin deacetylase proteinfamily and/or to a homolog of a SIRT1 protein. In one embodiment, aSIRT3 protein includes human SIRT3 (GenBank Accession No. AAH01042,NP_(—)036371, or NP_(—)001017524) and mouse SIRT3 (GenBank Accession No.NP_(—)071878) proteins, and equivalents and fragments thereof. Inanother embodiment, a SIRT3 protein includes a polypeptide comprising asequence consisting of, or consisting essentially of, the amino acidsequence set forth in GenBank Accession Nos. AAH01042, NP_(—)036371,NP_(—)001017524, or NP_(—)071878. SIRT3 proteins include polypeptidescomprising all or a portion of the amino acid sequence set forth inGenBank Accession AAH01042, NP_(—)036371, NP_(—)001017524, orNP_(—)071878; the amino acid sequence set forth in GenBank AccessionNos. AAH01042, NP_(—)036371, NP_(—)001017524, or NP_(—)071878 with 1 toabout 2, 3, 5, 7, 10, 15, 20, 30, 50, 75 or more conservative amino acidsubstitutions; an amino acid sequence that is at least 60%, 70%, 80%,90%, 95%, 96%, 97%, 98%, or 99% identical to GenBank Accession Nos.AAH01042, NP_(—)036371, NP_(—)001017524, or NP_(—)071878, and functionalfragments thereof. Polypeptides of the invention also include homologs(e.g., orthologs and paralogs), variants, or fragments, of GenBankAccession Nos. AAH01042, NP_(—)036371, NP_(—)001017524, or NP_(—)071878.In one embodiment, a SIRT3 protein includes a fragment of SIRT3 proteinthat is produced by cleavage with a mitochondrial matrix processingpeptidase (MPP) and/or a mitochondrial intermediate peptidase (MIP).

The terms “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” areart-recognized and refer to the administration of a subject composition,therapeutic or other material other than directly into the centralnervous system, such that it enters the patient's system and, thus, issubject to metabolism and other like processes.

The term “therapeutic agent” is art-recognized and refers to anychemical moiety that is a biologically, physiologically, orpharmacologically active substance that acts locally or systemically ina subject. The term also means any substance intended for use in thediagnosis, cure, mitigation, treatment or prevention of disease or inthe enhancement of desirable physical or mental development and/orconditions in an animal or human.

The term “therapeutic effect” is art-recognized and refers to a local orsystemic effect in animals, particularly mammals, and more particularlyhumans caused by a pharmacologically active substance. The phrase“therapeutically-effective amount” means that amount of such a substancethat produces some desired local or systemic effect at a reasonablebenefit/risk ratio applicable to any treatment. The therapeuticallyeffective amount of such substance will vary depending upon the subjectand disease condition being treated, the weight and age of the subject,the severity of the disease condition, the manner of administration andthe like, which can readily be determined by one of ordinary skill inthe art. For example, certain compositions described herein may beadministered in a sufficient amount to produce a desired effect at areasonable benefit/risk ratio applicable to such treatment.

“Treating” a condition or disease refers to curing as well asameliorating at least one symptom of the condition or disease.

The term “vision impairment” refers to diminished vision, which is oftenonly partially reversible or irreversible upon treatment (e.g.,surgery). Particularly severe vision impairment is termed “blindness” or“vision loss”, which refers to a complete loss of vision, vision worsethan 20/200 that cannot be improved with corrective lenses, or a visualfield of less than 20 degrees diameter (10 degrees radius).

2. Sirtuin Modulators

In one aspect, the invention provides novel sirtuin-modulating compoundsfor treating and/or preventing a wide variety of diseases and disordersincluding, for example, diseases or disorders related to aging orstress, diabetes, obesity, neurodegenerative diseases, ocular diseasesand disorders, cardiovascular disease, blood clotting disorders,inflammation, cancer, and/or flushing, etc. Sirtuin-modulating compoundsthat increase the level and/or activity of a sirtuin protein may also beused for treating a disease or disorder in a subject that would benefitfrom increased mitochondrial activity, for enhancing muscle performance,for increasing muscle ATP levels, or for treating or preventing muscletissue damage associated with hypoxia or ischemia. Other compoundsdisclosed herein may be suitable for use in a pharmaceutical compositionand/or one or more methods disclosed herein.

In one embodiment, sirtuin-modulating compounds of the invention arerepresented by Structural Formula (I):

or a salt thereof, wherein:

two of X¹ to X⁴ are selected from —CR*— and —N—;

the other two of X¹ to X⁴ are —CR*—;

R¹ is a solubilizing group;

R² is a phenyl group optionally substituted with a lower alkyl, loweralkoxy, halogen, nitrile or —CF₃, or R² is a 5- to 6-memberedheterocycle containing an N heteroatom and, optionally, a secondheteroatom selected from N, O or S, wherein said heterocycle isoptionally substituted with methyl or a halogen;

R* is independently selected at each occurrence from —H, lower alkyl orhalogen;

R is —H or —CH₃;

R′ is —CH₃ or a halogen; and

n is an integer from 0-4.

Typically, R is —H and n is 0, such that compounds of Structural Formula(I) are represented by Structural Formula (II):

or a salt thereof.

Preferred values in compounds of Structural Formula (I) and (II) are asfollows:

two of X¹ to X⁴ are selected from —CR*— and —N—;

the other two of X¹ to X⁴ are —CR*—;

R* is independently selected at each occurrence from —H, lower alkyl orhalogen;

R¹ is a solubilizing group; and

R² is selected from phenyl optionally substituted with one or moresubstituents independently selected from —CN, —F, —Cl and —CF₃, and wheneach of X¹ to X⁴ is —CR*—, R² is additionally selected from a 5- to6-membered heterocycle containing an N heteroatom and, optionally, asecond heteroatom selected from N, O or S, wherein said heterocycle isoptionally substituted with methyl.

In certain embodiments, each of X¹ to X⁴ is —CR*—. In other embodiments,one of X¹ to X⁴ is —N— and the remainder are —CR*—. In certainembodiments, two of X¹ to X⁴ are —N— and the remainder are —CR*—. Incertain embodiments, wherein two of X¹ to X⁴ are —N—, X¹ and X² are —N—.In certain embodiments, wherein two of X¹ to X⁴ are —N—, X¹ and X⁴ are—N—. In certain embodiments, when one of X¹ to X⁴ is —N—, X¹ is —N—. Incertain embodiments, R* is H.

In certain embodiments, such as when each of X¹ to X⁴ is —CR*—, R² isselected from phenyl, fluorophenyl, difluorophenyl, chlorophenyl,methylthiazolyl, pyrimidinyl, pyridyl and pyrazolyl. In certain suchembodiments, R² is selected from phenyl, fluorophenyl, difluorophenyl,chlorophenyl, 2-methylthiazol-4-yl, pyridyl and pyrazol-1-yl.Preferably, R² is phenyl or pyridyl.

In certain embodiments, R¹ is —CH₂—R³ and R³ is a nitrogen-containingheterocycle optionally substituted with one or more substituentsselected from C₁-C₄ alkyl, amino, halogen, methoxy andmethoxy-C₁-C₄-alkyl. In these embodiments, X¹ to X⁴ and R² can have anyof the values described above. In certain such embodiments, R² isphenyl, pyridyl or 3-fluorophenyl; X² and X³ are —CR*— and X¹ and X⁴ areindependently selected from —CR*— or —N—; or both.

In certain embodiments, R¹ is —CH₂—R³; and R³ is selected frompiperazin-1-yl, 4-(methoxyethyl-piperazin-1-yl,3,5-dimethylpiperazin-1-yl, morpholin-4-yl, piperidin-1-yl,4-aminopiperidin-1-yl, pyrrolidin-1-yl, 3-fluoropyrrolidin-1-yl,—NH-(pyrrolidin-3-yl), and 1,4-diaza-bicyclo[2.2.1]heptan-1-yl. In theseembodiments, X¹ to X⁴ and R² can have any of the values described above,but typically R² is phenyl, pyridyl or 3-fluorophenyl; X² and X³ are—CH— and X¹ and X⁴ are independently selected from —CH— or —N—; or both.

In certain such embodiments, R³ is selected from4-(methoxyethyl)-piperazin-1-yl, morpholin-4-yl, piperidin-1-yl and4-aminopiperidin-1-yl. When R³ has these values, R² is typically phenyl,3-fluorophenyl or pyridyl. Also, typically X² and X³ are —CH— and X¹ andX⁴ are independently selected from —CH— or —N—. In particularembodiments, X¹ and X⁴ are independently selected from —CH— or —N—; X²and X³ are —CH—; R² is phenyl, 3-fluorophenyl or pyridyl; and R¹ is—CH₂—R³ where R³ is selected from 4-(methoxyethyl)-piperazin-1-yl,morpholin-4-yl, piperidin-1-yl and 4-aminopiperidin-1-yl.

In certain embodiment, sirtuin-modulating compounds encompassed byStructural Formula (I) are represented by Structural Formula (III):

or a salt thereof, wherein:

one of X¹ to X³ is selected from —CH— and —N—;

the other two of X¹ to X³ are —CH—;

R¹ is a solubilizing group;

R² is a phenyl group optionally substituted with a methyl, halogen or—CF₃, or R² is a 5- to 6-membered heterocycle containing an N heteroatomand, optionally, a second heteroatom selected from N, O or S, whereinsaid heterocycle is optionally substituted with methyl or a halogen;

R is —H or —CH₃;

R′ is —CH₃ or a halogen; and

n is an integer from 0-4.

Typically, R is —H and n is 0, such that compounds of Structural Formula(III) are represented by Structural Formula (IV):

Preferred values in compounds of Structural Formula (III) and (IV) areas follows:

one of X¹ to X³ is selected from —CH— and —N—;

the other two of X¹ to X³ are —CH—;

R¹ is a solubilizing group; and

R² is selected from phenyl and fluorophenyl, and, when each of X¹ to X³is —CH—, R² is additionally selected from a 5- to 6-membered heterocyclecontaining an N heteroatom and, optionally, a second heteroatom selectedfrom N, O or S, wherein said heterocycle is optionally substituted withmethyl.

In certain embodiments, each of X¹ to X³ is —CH—. In other embodiments,one of X¹ to X³ is —N— and the remainder are —CH—. Typically, when oneof X¹ to X³ is —N—, X¹ is —N—.

In certain embodiments, such as when each of X¹ to X³ is —CH—, R² isselected from phenyl, fluorophenyl, methylthiazolyl, pyrimidinyl,pyridyl and pyrazolyl. In certain such embodiments, R² is selected fromphenyl, fluorophenyl, 2-methylthiazol-4-yl, pyridyl and pyrazol-1-yl.Preferably, R² is phenyl or pyridyl.

In certain embodiments, R¹ is —CH₂—R³ and R³ is a nitrogen-containingheterocycle optionally substituted with one or more substituentsselected from C₁-C₄ alkyl, amino, halogen, methoxy andmethoxy-C₁-C₄-alkyl. In these embodiments, X¹ to X³ and R² can have anyof the values described above. In certain such embodiments, R² isphenyl, pyridyl or 3-fluorophenyl; X² and X³ are —CH— and X¹ is —CH— or—N—; or both.

In certain embodiments, R¹ is —CH₂—R³; and R³ is selected frompiperazin-1-yl, 4-(methoxyethyl-piperazin-1-yl,3,5-dimethylpiperazin-1-yl, morpholin-4-yl, piperidin-1-yl,4-aminopiperidin-1-yl, pyrrolidin-1-yl, 3-fluoropyrrolidin-1-yl,—NH-(pyrrolidin-3-yl), and 1,4-diaza-bicyclo[2.2.1]heptan-1-yl. In theseembodiments, X¹ to X³ and R² can have any of the values described above,but typically R² is phenyl, pyridyl or 3-fluorophenyl; X² and X³ are—CH— and X¹ is —CH— or —N—; or both.

In certain such embodiments, R³ is selected from4-(methoxyethyl)-piperazin-1-yl, morpholin-4-yl, piperidin-1-yl and4-aminopiperidin-1-yl. When R³ has these values, R² is typically phenyl,3-fluorophenyl or pyridyl. Also, typically X² and X³ are —CH— and X¹ is—CH— or —N—. In particular embodiments, X¹ is —CH— or —N—; X² and X³ are—CH—; R² is phenyl, 3-fluorophenyl or pyridyl; and R¹ is —CH₂—R³ whereR³ is selected from 4-(methoxyethyl)-piperazin-1-yl, morpholin-4-yl,piperidin-1-yl and 4-aminopiperidin-1-yl.

In another embodiment, sirtuin-modulating compounds of the invention arerepresented by Structural Formula (V):

or a salt thereof, wherein:

ring A is selected from:

R¹ is a solubilizing group; and

R^(#) is a —H or —O—CH₃.

In yet another embodiment, sirtuin-modulating compounds of the inventionare represented by Structural Formula (VI):

or a salt thereof, wherein:

ring B is selected from:

R¹ is a solubilizing group.

Preferred solubilizing groups for Structural Formula (V)-(VI) are thesame as for Structural Formulas (I)-(IV) as described above.

Compounds of the invention, including novel compounds of the invention,can also be used in the methods described herein.

Sirtuin-modulating compounds of the invention advantageously modulatethe level and/or activity of a sirtuin protein, particularly thedeacetylase activity of the sirtuin protein.

Separately or in addition to the above properties, certainsirtuin-modulating compounds of the invention do not substantially haveone or more of the following activities: inhibition of PI3-kinase,inhibition of aldoreductase, inhibition of tyrosine kinase,transactivation of EGFR tyrosine kinase, coronary dilation, orspasmolytic activity, at concentrations of the compound that areeffective for modulating the deacetylation activity of a sirtuin protein(e.g., such as a SIRT1 and/or a SIRT3 protein).

An alkyl group is a straight chained, branched or cyclic non-aromatichydrocarbon which is completely saturated. Typically, a straight chainedor branched alkyl group has from 1 to about 20 carbon atoms, preferablyfrom 1 to about 10, and a cyclic alkyl group has from 3 to about 10carbon atoms, preferably from 3 to about 8. Examples of straight chainedand branched alkyl groups include methyl, ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl and octyl. A C₁-C₄straight chained or branched alkyl group is also referred to as a “loweralkyl” group.

Aromatic (aryl) groups include carbocyclic aromatic groups such asphenyl, naphthyl, and anthracyl, and heteroaryl groups such asimidazolyl, thienyl, furyl, pyridyl, pyrimidyl, pyranyl, pyrazolyl,pyrroyl, pyrazinyl, thiazolyl, oxazolyl, and tetrazolyl.

Aromatic groups also include fused polycyclic aromatic ring systems inwhich a carbocyclic aromatic ring or heteroaryl ring is fused to one ormore other heteroaryl rings. Examples include benzothienyl, benzofuryl,indolyl, quinolinyl, benzothiazole, benzoxazole, benzimidazole,quinolinyl, isoquinolinyl and isoindolyl.

Suitable substituents on an alkyl or aryl group (carbocyclic andheteroaryl) are those which do not substantially interfere with theability of the disclosed compounds to have one or more of the propertiesdisclosed herein. A substituent substantially interferes with theproperties of a compound when the magnitude of the property is reducedby more than about 50% in a compound with the substituent compared witha compound without the substituent. Examples of suitable substituentsinclude —OH, halogen (—Br, —Cl, —I and —F), —OR^(a), —O—COR^(a),—COR^(a), —C(O)R^(a), —CN, —NO², —COOH, —COOR^(a), —OCO₂R^(a),—C(O)NR^(a)R^(b), —OC(O)NR^(a)R^(b), —SO₃H, —NH₂, —NHR^(a),—N(R^(a)R^(b)), —COOR^(a), —CHO, —CONH₂, —CONHR^(a), —CON(R^(a)R^(b)),—NHCOR^(a), —NRCOR^(a), —NHCONH₂, —NHCONR^(a)H, —NHCON(R^(a)R^(b)),—NR^(c)CONH₂, —NR^(c)CONR^(a)H, —NR^(c)CON(R^(a)R^(b)), —C(═NH)—NH₂,—C(═NH)—NHR^(a), —C(═NH)—N(R^(a)R^(b)), —C(═NR^(c))—NH₂,—C(═NR^(c))—NHR^(a), —C(═NR^(c))—N(R^(a)R^(b)), —NH—C(═NH)—NH₂,—NH—C(═NH)—NHR^(a), —NH—C(═NH)—N(R^(a)R^(b)), —NH—C(═NR^(c))—NH₂,—NH—C(═NR^(c))—NHR^(a), —NH—C(═NR^(c))—N(R^(a)R^(b)),—NR^(d)H—C(═NH)—NH₂, —NR^(d)—C(═NH)—NHR^(a),—NR^(d)—C(═NH)—N(R^(a)R^(b)), —NR^(d)—C(═NR^(c))—NH₂,—NR^(d)—C(═NR^(c))—NHR^(a), —NR^(d)—C(═NR^(c))—N(R^(a)R^(b)), —NHNH₂,—NHNHR^(a), —NHR^(a)R^(b), —SO₂NH₂, —SO₂NHR_(a), —SO₂NR^(a)R^(b),—CH═CHR^(a), —CH═CR^(a)R^(b), —CR^(c)═CR^(a)R^(b), CR^(c)═CHR^(a),CR^(c)═CR^(a)R^(b), —CCR^(a), —SH, —SO_(k)R^(a) (k is 0, 1 or 2),—S(O)_(k)OR^(a) (k is 0, 1 or 2) and —NH—C(═NH)—NH₂. R^(a)-R^(d) areeach independently an optionally substituted group selected from analiphatic, benzyl, or aromatic group, preferably an alkyl, benzylic oraryl group. Optional substituents on R^(a)-R^(d) are selected from NH₂,NH(C₁₋₄aliphatic), N(C₁₋₄aliphatic)₂, halogen, C₁₋₄aliphatic, OH,O(C₁₋₄aliphatic), NO₂, CN, CO₂H, CO₂(C₁₋₄aliphatic), O(haloC₁₋₄aliphatic), or haloC₁₋₄aliphatic, wherein each of the foregoingC₁₋₄aliphatic groups of is unsubstituted. In addition, —NR^(a)R^(b),taken together, can also form a substituted or unsubstitutednon-aromatic heterocyclic group. A substituted aliphatic or substitutedaryl group can have more than one substituent.

Typical substituents on an aryl ring are selected from a solubilizinggroup, halogen; —R^(o); —OR^(o); —SR^(o); 1,2-methylenedioxy;1,2-ethylenedioxy; phenyl (Ph) optionally substituted with R^(o); —O(Ph)optionally substituted with R^(o); —(CH₂)₁₋₂(Ph), optionally substitutedwith R^(o); —CH═CH(Ph), optionally substituted with R^(o); —NO₂; —CN;—N(R^(o))₂; —C(O)C(O)R^(o); —C(O)CH₂C(O)R^(o); —CO₂R^(o); —C(O)R^(o);—S(O)₂R^(o); —SO₂N(R^(o))₂; —S(O)R^(o); —NR^(o)SO₂N(R^(o))₂;—NR^(o)SO₂R^(o); —C(═S)N(R^(o))₂; or —C(═NH)—N(R^(o))₂; or wherein eachindependent occurrence of R^(o) is selected from hydrogen, optionallysubstituted C₁₋₆ aliphatic, an unsubstituted 5-6 membered heteroaryl orheterocyclic ring, phenyl, —O(Ph), or —CH₂(Ph), or, notwithstanding thedefinition above, two independent occurrences of R^(o), on the samesubstituent or different substituents, taken together with the atom(s)to which each R^(o) group is bound, form a 3-8-membered cycloalkyl,heterocyclyl, aryl, or heteroaryl ring having 0-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. Optionalsubstituents on the aliphatic group of R^(o) are selected from NH₂,NH(C₁₋₄aliphatic), N(C₁₋₄aliphatic)₂, halogen, C₁₋₄aliphatic, OH,O(C₁₋₄aliphatic), NO₂, CN, CO₂H, CO₂(C₁₋₄aliphatic), O(haloC₁₋₄aliphatic), or haloC₁₋₄aliphatic, wherein each of the foregoingC₁₋₄aliphatic groups of R^(o) is unsubstituted

Combinations of substituents and variables envisioned by this inventionare only those that result in the formation of stable compounds. As usedherein, the term “stable” refers to compounds that possess stabilitysufficient to allow manufacture and that maintain the integrity of thecompound for a sufficient period of time to be useful for the purposesdetailed herein.

As used herein, a “solubilizing group” is a moiety that has hydrophiliccharacter sufficient to improve or increase the water-solubility of thecompound in which it is included, as compared to an analog compound thatdoes not include the group. The hydrophilic character can be achieved byany means, such as by the inclusion of functional groups that ionizeunder the conditions of use to form charged moieties (e.g., carboxylicacids, sulfonic acids, phosphoric acids, amines, etc.); groups thatinclude permanent charges (e.g., quaternary ammonium groups); and/orheteroatoms or heteroatomic groups (e.g., O, S, N, NH,N—(CH₂)_(y)—R^(a), N—(CH₂)_(y)—C(O)R^(a), N—(CH₂)_(y)—C(O)OR^(a),N—(CH₂)_(y)—S(O)₂R^(a)—, N—(CH₂)_(y)—S(O)₂OR^(a),N—(CH₂)_(y)—C(O)NR^(a)R^(a), etc., wherein R^(a) is selected fromhydrogen, lower alkyl, lower cycloalkyl, (C6-C14) aryl, phenyl,naphthyl, (C7-C20) arylalkyl and benzyl, wherein R^(a) is optionallysubstituted; and y is an integer ranging from 0 to 6), optionallysubstituted heterocyclic groups (e.g., —(CH₂)_(n)—R^(b),—(CH₂)_(n)—C(O)—R^(b), —(CH₂)_(n)—O—(CH₂)_(n)—R^(b), wherein R^(b) isselected from an optionally substituted saturated monocyclicheterocycle, an optionally substituted saturated bicyclic fusedheterocycle, an optionally substituted saturated bicyclic spiroheterocycle, an optionally substituted heteroaryl and an optionallysubstituted partially substituted non-aryl heterocycle; and n is aninteger ranging from 0 to 2). It should be understood that substituentspresent on R^(a) or R^(b) need not improve or increase water solubilityover their unsubstituted counterparts to be within the scope of thisdefinition. All that is required is that such substituents do notsignificantly reverse the improvement in water-solubility afforded bythe unsubstituted R^(a) or R^(b) moiety.

In one embodiment, the solubilizing group increases the water-solubilityof the corresponding compound lacking the solubilizing group at least5-fold, preferably at least 10-fold, more preferably at least 20-foldand most preferably at least 50-fold.

In one preferred embodiment, the solubilizing group is a moiety of theformula: —(CH₂)_(n)—R¹⁰⁰—N(R¹⁰¹)(R¹⁰¹), wherein:

n is selected from 0, 1 or 2;R¹⁰⁰ is selected from a bond, —C(O)—, or —O(CH₂)_(n); andeach R¹⁰¹ is independently selected from:

-   -   a. hydrogen;    -   b. C₁-C₄ straight or branched alkyl, wherein said alkyl is        optionally substituted with halo, CN, OH, O—(C₁-C₄ straight or        branched alkyl), N(R₁′)(R₁′), or ═O;

-   -   f. both R¹⁰¹ moieties are taken together with the nitrogen atom        to which they are bound to form a ring of the structure

-   -   g. both R¹⁰¹ moieties are taken together with the nitrogen atom        to which they are bound to form a 5-membered heteroaryl ring        containing 1 to 3 additional N atoms, wherein said heteroaryl        ring is optionally substituted with R₁′;        wherein:

each Z is independently selected from —O—, —S—, —NR₁′—, or—C(R⁵⁰)(R⁵⁰)—, wherein:

-   -   at least three of Z₂₀, Z₂₁, Z₂₂, and Z₂₃ are —C(R⁵⁰)(R⁵⁰)—;    -   at least three of Z₂₄, Z₂₅, Z₂₆, Z₂₇, and Z₂₈ are —C(R⁵⁰)(R⁵⁰)—;    -   at least four of Z₃₀, Z₃₁, Z₃₂, and Z₃₃ are —C(R⁵⁰)(R⁵⁰)—; and    -   at least four of Z₃₄, Z₃₅, Z₃₆, Z₃₇, and Z₃₈ are —C(R⁵⁰)(R⁵⁰)—;

each R₁′ is independently selected from hydrogen or a C₁-C₃ straight orbranched alkyl optionally substituted with one or more substituentindependently selected from halo, —CN, —OH, —OCH₃, —NH₂, —NH(CH₃),—N(CH₃)₂, or ═O;

each R⁵⁰ is independently selected from R₁′, halo, CN, OH, O—(C₁-C₄straight or branched alkyl), N(R₁′)(R₁′), ═CR₁′, SR₁′, ═NR₁′, ═NOR₁′, or═O;

any two suitable non-cyclic R⁵⁰ are optionally bound to one anotherdirectly or via a C₁ to C₂ alkylene, alkenylene or alkanediylidenebridge to produce a bicyclic fused or spiro ring; and

ring structure is optionally benzofused or fused to a monocyclicheteroaryl to produce a bicyclic ring.

For clarity, the term “C₁ to C₂ alkylene, alkenylene or alkanediylidenebridge” means the multivalent structures —CH₂—, —CH₂—CH₂—, —CH═, ═CH—,—CH═CH—, or ═CH—CH═. The two R⁵⁰ moieties that are optionally bound toone another can be either on the same carbon atom or different carbonatoms. The former produces a spiro bicyclic ring, while the latterproduces a fused bicyclic ring. It will be obvious to those of skill inthe art that when two R⁵⁰ are bound to one another to form a ring(whether directly or through one of the recited bridges), one or moreterminal hydrogen atoms on each R⁵⁰ will be lost. Accordingly, a“suitable non-cyclic R⁵⁰” moiety available for forming a ring is anon-cyclic R⁵⁰ that comprises at least one terminal hydrogen atom.

In another embodiment, the solubilizing group is a moiety of theformula: —(CH₂)_(n)—O—R¹⁰¹, wherein n and R¹⁰¹ are as defined above.

In yet another embodiment, the solubilizing group is a moiety of theformula: —(CH₂)_(n)—C(O)—R₁′, wherein n and R₁′ are as defined above.

In certain embodiments, a solubilizing group is selected from—(CH₂)_(n)—R¹⁰², wherein n is 0, 1 or 2, preferably 2; and R¹⁰² isselected from

wherein R₁′ groups are as defined above.

In certain particular embodiments, a solubilizing group is selected from2-dimethylaminoethylcarbamoyl, piperazin-1-ylcarbonyl,piperazinylmethyl, dimethylaminomethyl, 4-methylpiperazin-1-ylmethyl,4-aminopiperidin-1-yl-methyl, 4-fluoropiperidin-1-yl-methyl,morpholinomethyl, pyrrolidin-1-ylmethyl,2-oxo-4-benzylpiperazin-1-ylmethyl, 4-benzylpiperazin-1-ylmethyl,3-oxopiperazin-1-ylmethyl, piperidin-1-ylmethyl, piperazin-1-ylethyl,2,3-dioxopropylaminomethyl, thiazolidin-3-ylmethyl,4-acetylpiperazin-1-ylmethyl, 4-acetylpiperazin-1-yl, morpholino,3,3-difluoroazetidin-1-ylmethyl, 2H-tetrazol-5-ylmethyl,thiomorpholin-4-ylmethyl, 1-oxothiomorpholin-4-ylmethyl,1,1-dioxothiomorpholin-4-ylmethyl, 1H-imidazol-1-ylmethyl,3,5-dimethylpiperazin-1 ylmethyl, 4-hydroxypiperidin-1-ylmethyl,N-methyl(1-acetylpiperidin-4-yl)-aminomethyl,N-methylquinuclidin-3-ylaminomethyl, 1H-1,2,4-triazol-1-ylmethyl,1-methylpiperidin-3-yl-oxymethyl, or 4-fluoropiperidin-1-yl.

To the extent not included within any of the definitions set forthabove, the term “solubilizing group” also includes moieties disclosed asbeing attached to the 7-position of1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid(ciprofloxacin) and its derivatives, as disclosed in PCT publications WO2005/026165, WO 2005/049602, and WO 2005/033108, and European Patentpublications EP 0343524, EP 0688772, EP 0153163, EP 0159174; as well as“water-solubilizing groups” described in United States patentpublication 2006/0035891. The disclosure of each of these patentpublications is incorporated herein by reference.

The compounds disclosed herein also include partially and fullydeuterated variants. In certain embodiments, one or more deuterium atomsare present for kinetic studies. One of ordinary skill in the art canselect the sites at which such deuterium atoms are present.

Also included in the present invention are salts, particularlypharmaceutically acceptable salts, of the sirtuin-modulating compoundsdescribed herein. The compounds of the present invention that possess asufficiently acidic, a sufficiently basic, or both functional groups,can react with any of a number of inorganic bases, and inorganic andorganic acids, to form a salt. Alternatively, compounds that areinherently charged, such as those with a quaternary nitrogen, can form asalt with an appropriate counterion (e.g., a halide such as bromide,chloride, or fluoride, particularly bromide).

The compounds and salts thereof described herein also include thehydrates (e.g., hemihydrate, monohydrate, dihydrate, trihydrate,tetrahydrate) and solvates of the compounds and salts thereof. Suitablesolvents for preparation of solvates and hydrates can generally beselected by a skilled artisan.

The compounds and salts thereof can be present in amorphous orcrystalline (including co-crystalline and polymorph) forms.

Acids commonly employed to form acid addition salts are inorganic acidssuch as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuricacid, phosphoric acid, and the like, and organic acids such asp-toluenesulfonic acid, methanesulfonic acid, oxalic acid,p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, acetic acid, and the like. Examples of such salts includethe sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caproate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate,phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,mandelate, and the like.

Base addition salts include those derived from inorganic bases, such asammonium or alkali or alkaline earth metal hydroxides, carbonates,bicarbonates, and the like. Such bases useful in preparing the salts ofthis invention thus include sodium hydroxide, potassium hydroxide,ammonium hydroxide, potassium carbonate, and the like.

According to another embodiment, the present invention provides methodsof producing the above-defined sirtuin-modulating compounds. Thecompounds may be synthesized using conventional techniques.Advantageously, these compounds are conveniently synthesized fromreadily available starting materials.

One method of preparing compounds of the invention involves reacting thefollowing compound:

wherein X is a leaving group (e.g., a halogen such as Cl), with aring-closing agent (e.g., agent) to form the following compound:

This compound is typically chlorinated or brominated (e.g., withN-bromosuccinimide) to form the following compound:

where Hal represents a halogen, although other leaving groups areacceptable. This compound is then reacted with an optionally protectednitrogen-containing heteroring (e.g., an optionally protected R³ group)to form the following compound:

wherein the nitrogen-containing heteroring is optionally substituted andoptionally includes one or more additional heteroatoms. The compound isreduced with an appropriate reducing agent (e.g., iron powder) to formthe following compound:

This compound is subsequently reacted with a R²-substituted pyridinecarboxylic acid or a R²-substituted benzoic acid and, if needed,deprotected, to form a compound of the invention.

Synthetic chemistry transformations and methodologies useful insynthesizing the sirtuin-modulating compounds described herein are knownin the art and include, for example, those described in R. Larock,Comprehensive Organic Transformations (1989); T. W. Greene and P. G. M.Wuts, Protective Groups in Organic Synthesis, 2d. Ed. (1991); L. Fieserand M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis(1994); and L. Paquette, ed., Encyclopedia of Reagents for OrganicSynthesis (1995).

In an exemplary embodiment, a sirtuin-modulating compound may traversethe cytoplasmic membrane of a cell. For example, a compound may have acell-permeability of at least about 20%, 50%, 75%, 80%, 90% or 95%.

Sirtuin-modulating compounds described herein may also have one or moreof the following characteristics: the compound may be essentiallynon-toxic to a cell or subject; the sirtuin-modulating compound may bean organic molecule or a small molecule of 2000 amu or less, 1000 amu orless; a compound may have a half-life under normal atmosphericconditions of at least about 30 days, 60 days, 120 days, 6 months or 1year; the compound may have a half-life in solution of at least about 30days, 60 days, 120 days, 6 months or 1 year; a sirtuin-modulatingcompound may be more stable in solution than resveratrol by at least afactor of about 50%, 2 fold, 5 fold, 10 fold, 30 fold, 50 fold or 100fold; a sirtuin-modulating compound may promote deacetylation of the DNArepair factor Ku70; a sirtuin-modulating compound may promotedeacetylation of RelA/p65; a compound may increase general turnoverrates and enhance the sensitivity of cells to TNF-induced apoptosis.

In certain embodiments, a sirtuin-modulating compound does not have anysubstantial ability to inhibit a histone deacetylase (HDACs) class I, aHDAC class II, or HDACs I and II, at concentrations (e.g., in vivo)effective for modulating the deacetylase activity of the sirtuin. Forinstance, in preferred embodiments the sirtuin-modulating compound is asirtuin-activating compound and is chosen to have an EC₅₀ for activatingsirtuin deacetylase activity that is at least 5 fold less than the EC₅₀for inhibition of an HDAC I and/or HDAC II, and even more preferably atleast 10 fold, 100 fold or even 1000 fold less. Methods for assayingHDAC I and/or HDAC II activity are well known in the art and kits toperform such assays may be purchased commercially. See e.g., BioVision,Inc. (Mountain View, Calif.; world wide web at biovision.com) and ThomasScientific (Swedesboro, N.J.; world wide web at tomassci.com).

In certain embodiments, a sirtuin-modulating compound does not have anysubstantial ability to modulate sirtuin homologs. In one embodiment, anactivator of a human sirtuin protein may not have any substantialability to activate a sirtuin protein from lower eukaryotes,particularly yeast or human pathogens, at concentrations (e.g., in vivo)effective for activating the deacetylase activity of human sirtuin. Forexample, a sirtuin-activating compound may be chosen to have an EC₅₀ foractivating a human sirtuin, such as SIRT1 and/or SIRT3, deacetylaseactivity that is at least 5 fold less than the EC₅₀ for activating ayeast sirtuin, such as Sir2 (such as Candida, S. cerevisiae, etc.), andeven more preferably at least 10 fold, 100 fold or even 1000 fold less.In another embodiment, an inhibitor of a sirtuin protein from lowereukaryotes, particularly yeast or human pathogens, does not have anysubstantial ability to inhibit a sirtuin protein from humans atconcentrations (e.g., in vivo) effective for inhibiting the deacetylaseactivity of a sirtuin protein from a lower eukaryote. For example, asirtuin-inhibiting compound may be chosen to have an IC₅₀ for inhibitinga human sirtuin, such as SIRT1 and/or SIRT3, deacetylase activity thatis at least 5 fold less than the IC₅₀ for inhibiting a yeast sirtuin,such as Sir2 (such as Candida, S. cerevisiae, etc.), and even morepreferably at least 10 fold, 100 fold or even 1000 fold less.

In certain embodiments, a sirtuin-modulating compound may have theability to modulate one or more sirtuin protein homologs, such as, forexample, one or more of human SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6,or SIRT7. In one embodiment, a sirtuin-modulating compound has theability to modulate both a SIRT1 and a SIRT3 protein.

In other embodiments, a SIRT1 modulator does not have any substantialability to modulate other sirtuin protein homologs, such as, forexample, one or more of human SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, orSIRT7, at concentrations (e.g., in vivo) effective for modulating thedeacetylase activity of human SIRT1. For example, a sirtuin-modulatingcompound may be chosen to have an ED₅₀ for modulating human SIRT1deacetylase activity that is at least 5 fold less than the ED₅₀ formodulating one or more of human SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, orSIRT7, and even more preferably at least 10 fold, 100 fold or even 1000fold less. In one embodiment, a SIRT1 modulator does not have anysubstantial ability to modulate a SIRT3 protein.

In other embodiments, a SIRT3 modulator does not have any substantialability to modulate other sirtuin protein homologs, such as, forexample, one or more of human SIRT1, SIRT2, SIRT4, SIRT5, SIRT6, orSIRT7, at concentrations (e.g., in vivo) effective for modulating thedeacetylase activity of human SIRT3. For example, a sirtuin-modulatingcompound may be chosen to have an ED₅₀ for modulating human SIRT3deacetylase activity that is at least 5 fold less than the ED₅₀ formodulating one or more of human SIRT1, SIRT2, SIRT4, SIRT5, SIRT6, orSIRT7, and even more preferably at least 10 fold, 100 fold or even 1000fold less. In one embodiment, a SIRT3 modulator does not have anysubstantial ability to modulate a SIRT1 protein.

In certain embodiments, a sirtuin-modulating compound may have a bindingaffinity for a sirtuin protein of about 10⁻⁹M, 10⁻¹⁰M, 10⁻¹¹M, 10⁻¹²M orless. A sirtuin-modulating compound may reduce (activator) or increase(inhibitor) the apparent Km of a sirtuin protein for its substrate orNAD+ (or other cofactor) by a factor of at least about 2, 3, 4, 5, 10,20, 30, 50 or 100. In certain embodiments, Km values are determinedusing the mass spectrometry assay described herein. Preferred activatingcompounds reduce the Km of a sirtuin for its substrate or cofactor to agreater extent than caused by resveratrol at a similar concentration orreduce the Km of a sirtuin for its substrate or cofactor similar to thatcaused by resveratrol at a lower concentration. A sirtuin-modulatingcompound may increase the Vmax of a sirtuin protein by a factor of atleast about 2, 3, 4, 5, 10, 20, 30, 50 or 100. A sirtuin-modulatingcompound may have an ED50 for modulating the deacetylase activity of aSIRT1 and/or SIRT3 protein of less than about 1 nM, less than about 10nM, less than about 100 nM, less than about 1 μM, less than about 10 μM,less than about 100 μM, or from about 1-10 nM, from about 10-100 nM,from about 0.1-1 μM, from about 1-10 μM or from about 10-100 μM. Asirtuin-modulating compound may modulate the deacetylase activity of aSIRT1 and/or SIRT3 protein by a factor of at least about 5, 10, 20, 30,50, or 100, as measured in a cellular assay or in a cell based assay. Asirtuin-activating compound may cause at least about 10%, 30%, 50%, 80%,2 fold, 5 fold, 10 fold, 50 fold or 100 fold greater induction of thedeacetylase activity of a sirtuin protein relative to the sameconcentration of resveratrol. A sirtuin-modulating compound may have anED50 for modulating SIRT5 that is at least about 10 fold, 20 fold, 30fold, 50 fold greater than that for modulating SIRT1 and/or SIRT3.

3. Exemplary Uses

In certain aspects, the invention provides methods for modulating thelevel and/or activity of a sirtuin protein and methods of use thereof.

In certain embodiments, the invention provides methods for usingsirtuin-modulating compounds wherein the sirtuin-modulating compoundsactivate a sirtuin protein, e.g., increase the level and/or activity ofa sirtuin protein. Sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be useful for a variety oftherapeutic applications including, for example, increasing the lifespanof a cell, and treating and/or preventing a wide variety of diseases anddisorders including, for example, diseases or disorders related to agingor stress, diabetes, obesity, neurodegenerative diseases, cardiovasculardisease, blood clotting disorders, inflammation, cancer, and/orflushing, etc. The methods comprise administering to a subject in needthereof a pharmaceutically effective amount of a sirtuin-modulatingcompound, e.g., a sirtuin-activating compound.

While Applicants do not wish to be bound by theory, it is believed thatactivators of the instant invention may interact with a sirtuin at thesame location within the sirtuin protein (e.g., active site or siteaffecting the Km or Vmax of the active site). It is believed that thisis the reason why certain classes of sirtuin activators and inhibitorscan have substantial structural similarity.

In certain embodiments, the sirtuin-modulating compounds describedherein may be taken alone or in combination with other compounds. In oneembodiment, a mixture of two or more sirtuin-modulating compounds may beadministered to a subject in need thereof. In another embodiment, asirtuin-modulating compound that increases the level and/or activity ofa sirtuin protein may be administered with one or more of the followingcompounds: resveratrol, butein, fisetin, piceatannol, or quercetin. Inan exemplary embodiment, a sirtuin-modulating compound that increasesthe level and/or activity of a sirtuin protein may be administered incombination with nicotinic acid. In another embodiment, asirtuin-modulating compound that decreases the level and/or activity ofa sirtuin protein may be administered with one or more of the followingcompounds: nicotinamide (NAM), suranim; NF023 (a G-protein antagonist);NF279 (a purinergic receptor antagonist); Trolox(6-hydroxy-2,5,7,8,tetramethylchroman-2-carboxylic acid);(−)-epigallocatechin (hydroxy on sites 3,5,7,3′,4′,5′);(−)-epigallocatechin gallate (Hydroxy sites 5,7,3′,4′,5′ and gallateester on 3); cyanidin chloride (3,5,7,3′,4′-pentahydroxyflavyliumchloride); delphinidin chloride (3,5,7,3′,4′,5′-hexahydroxyflavyliumchloride); myricetin (cannabiscetin; 3,5,7,3′,4′,5′-hexahydroxyflavone);3,7,3′,4′,5′-pentahydroxyflavone; gossypetin(3,5,7,8,3′,4′-hexahydroxyflavone), sirtinol; and splitomicin. In yetanother embodiment, one or more sirtuin-modulating compounds may beadministered with one or more therapeutic agents for the treatment orprevention of various diseases, including, for example, cancer,diabetes, neurodegenerative diseases, cardiovascular disease, bloodclotting, inflammation, flushing, obesity, ageing, stress, etc. Invarious embodiments, combination therapies comprising asirtuin-modulating compound may refer to (1) pharmaceutical compositionsthat comprise one or more sirtuin-modulating compounds in combinationwith one or more therapeutic agents (e.g., one or more therapeuticagents described herein); and (2) co-administration of one or moresirtuin-modulating compounds with one or more therapeutic agents whereinthe sirtuin-modulating compound and therapeutic agent have not beenformulated in the same compositions (but may be present within the samekit or package, such as a blister pack or other multi-chamber package;connected, separately sealed containers (e.g., foil pouches) that can beseparated by the user; or a kit where the sirtuin modulating compound(s)and other therapeutic agent(s) are in separate vessels). When usingseparate formulations, the sirtuin-modulating compound may beadministered at the same, intermittent, staggered, prior to, subsequentto, or combinations thereof, with the administration of anothertherapeutic agent.

In certain embodiments, methods for reducing, preventing or treatingdiseases or disorders using a sirtuin-modulating compound may alsocomprise increasing the protein level of a sirtuin, such as human SIRT1,SIRT2 and/or SIRT3, or homologs thereof. Increasing protein levels canbe achieved by introducing into a cell one or more copies of a nucleicacid that encodes a sirtuin. For example, the level of a sirtuin can beincreased in a mammalian cell by introducing into the mammalian cell anucleic acid encoding the sirtuin, e.g., increasing the level of SIRT1by introducing a nucleic acid encoding the amino acid sequence set forthin GenBank Accession No. NP_(—)036370 and/or increasing the level ofSIRT3 by introducing a nucleic acid encoding the amino acid sequence setforth in GenBank Accession No. AAH10042.

A nucleic acid that is introduced into a cell to increase the proteinlevel of a sirtuin may encode a protein that is at least about 80%, 85%,90%, 95%, 98%, or 99% identical to the sequence of a sirtuin, e.g.,SIRT1 and/or SIRT3 protein. For example, the nucleic acid encoding theprotein may be at least about 80%, 85%, 90%, 95%, 98%, or 99% identicalto a nucleic acid encoding a SIRT1 (e.g. GenBank Accession No.NM_(—)012238) and/or SIRT3 (e.g., GenBank Accession No. BC001042)protein. The nucleic acid may also be a nucleic acid that hybridizes,preferably under stringent hybridization conditions, to a nucleic acidencoding a wild-type sirtuin, e.g., SIRT1 and/or SIRT3 protein.Stringent hybridization conditions may include hybridization and a washin 0.2×SSC at 65° C. When using a nucleic acid that encodes a proteinthat is different from a wild-type sirtuin protein, such as a proteinthat is a fragment of a wild-type sirtuin, the protein is preferablybiologically active, e.g., is capable of deacetylation. It is onlynecessary to express in a cell a portion of the sirtuin that isbiologically active. For example, a protein that differs from wild-typeSIRT1 having GenBank Accession No. NP_(—)036370, preferably contains thecore structure thereof. The core structure sometimes refers to aminoacids 62-293 of GenBank Accession No. NP_(—)036370, which are encoded bynucleotides 237 to 932 of GenBank Accession No. NM_(—)012238, whichencompasses the NAD binding as well as the substrate binding domains.The core domain of SIRT1 may also refer to about amino acids 261 to 447of GenBank Accession No. NP_(—)036370, which are encoded by nucleotides834 to 1394 of GenBank Accession No. NM_(—)012238; to about amino acids242 to 493 of GenBank Accession No. NP_(—)036370, which are encoded bynucleotides 777 to 1532 of GenBank Accession No. NM_(—)012238; or toabout amino acids 254 to 495 of GenBank Accession No. NP_(—)036370,which are encoded by nucleotides 813 to 1538 of GenBank Accession No.NM_(—)012238. Whether a protein retains a biological function, e.g.,deacetylation capabilities, can be determined according to methods knownin the art.

In certain embodiments, methods for reducing, preventing or treatingdiseases or disorders using a sirtuin-modulating compound may alsocomprise decreasing the protein level of a sirtuin, such as human SIRT1,SIRT2 and/or SIRT3, or homologs thereof. Decreasing a sirtuin proteinlevel can be achieved according to methods known in the art. Forexample, an siRNA, an antisense nucleic acid, or a ribozyme targeted tothe sirtuin can be expressed in the cell. A dominant negative sirtuinmutant, e.g., a mutant that is not capable of deacetylating, may also beused. For example, mutant H363Y of SIRT1, described, e.g., in Luo et al.(2001) Cell 107:137 can be used. Alternatively, agents that inhibittranscription can be used.

Methods for modulating sirtuin protein levels also include methods formodulating the transcription of genes encoding sirtuins, methods forstabilizing/destabilizing the corresponding mRNAs, and other methodsknown in the art.

Aging/Stress

In one embodiment, the invention provides a method extending thelifespan of a cell, extending the proliferative capacity of a cell,slowing aging of a cell, promoting the survival of a cell, delayingcellular senescence in a cell, mimicking the effects of calorierestriction, increasing the resistance of a cell to stress, orpreventing apoptosis of a cell, by contacting the cell with asirtuin-modulating compound of the invention that increases the leveland/or activity of a sirtuin protein. In an exemplary embodiment, themethods comprise contacting the cell with a sirtuin-activating compound.

The methods described herein may be used to increase the amount of timethat cells, particularly primary cells (i.e., cells obtained from anorganism, e.g., a human), may be kept alive in a cell culture. Embryonicstem (ES) cells and pluripotent cells, and cells differentiatedtherefrom, may also be treated with a sirtuin-modulating compound thatincreases the level and/or activity of a sirtuin protein to keep thecells, or progeny thereof, in culture for longer periods of time. Suchcells can also be used for transplantation into a subject, e.g., afterex vivo modification.

In one embodiment, cells that are intended to be preserved for longperiods of time may be treated with a sirtuin-modulating compound thatincreases the level and/or activity of a sirtuin protein. The cells maybe in suspension (e.g., blood cells, serum, biological growth media,etc.) or in tissues or organs. For example, blood collected from anindividual for purposes of transfusion may be treated with asirtuin-modulating compound that increases the level and/or activity ofa sirtuin protein to preserve the blood cells for longer periods oftime. Additionally, blood to be used for forensic purposes may also bepreserved using a sirtuin-modulating compound that increases the leveland/or activity of a sirtuin protein. Other cells that may be treated toextend their lifespan or protect against apoptosis include cells forconsumption, e.g., cells from non-human mammals (such as meat) or plantcells (such as vegetables).

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may also be applied during developmental and growthphases in mammals, plants, insects or microorganisms, in order to, e.g.,alter, retard or accelerate the developmental and/or growth process.

In another embodiment, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used to treat cellsuseful for transplantation or cell therapy, including, for example,solid tissue grafts, organ transplants, cell suspensions, stem cells,bone marrow cells, etc. The cells or tissue may be an autograft, anallograft, a syngraft or a xenograft. The cells or tissue may be treatedwith the sirtuin-modulating compound prior toadministration/implantation, concurrently withadministration/implantation, and/or post administration/implantationinto a subject. The cells or tissue may be treated prior to removal ofthe cells from the donor individual, ex vivo after removal of the cellsor tissue from the donor individual, or post implantation into therecipient. For example, the donor or recipient individual may be treatedsystemically with a sirtuin-modulating compound or may have a subset ofcells/tissue treated locally with a sirtuin-modulating compound thatincreases the level and/or activity of a sirtuin protein. In certainembodiments, the cells or tissue (or donor/recipient individuals) mayadditionally be treated with another therapeutic agent useful forprolonging graft survival, such as, for example, an immunosuppressiveagent, a cytokine, an angiogenic factor, etc.

In yet other embodiments, cells may be treated with a sirtuin-modulatingcompound that increases the level and/or activity of a sirtuin proteinin vivo, e.g., to increase their lifespan or prevent apoptosis. Forexample, skin can be protected from aging (e.g., developing wrinkles,loss of elasticity, etc.) by treating skin or epithelial cells with asirtuin-modulating compound that increases the level and/or activity ofa sirtuin protein. In an exemplary embodiment, skin is contacted with apharmaceutical or cosmetic composition comprising a sirtuin-modulatingcompound that increases the level and/or activity of a sirtuin protein.Exemplary skin afflictions or skin conditions that may be treated inaccordance with the methods described herein include disorders ordiseases associated with or caused by inflammation, sun damage ornatural aging. For example, the compositions find utility in theprevention or treatment of contact dermatitis (including irritantcontact dermatitis and allergic contact dermatitis), atopic dermatitis(also known as allergic eczema), actinic keratosis, keratinizationdisorders (including eczema), epidermolysis bullosa diseases (includingpenfigus), exfoliative dermatitis, seborrheic dermatitis, erythemas(including erythema multiforme and erythema nodosum), damage caused bythe sun or other light sources, discoid lupus erythematosus,dermatomyositis, psoriasis, skin cancer and the effects of naturalaging. In another embodiment, sirtuin-modulating compounds that increasethe level and/or activity of a sirtuin protein may be used for thetreatment of wounds and/or burns to promote healing, including, forexample, first-, second- or third-degree burns and/or a thermal,chemical or electrical burns. The formulations may be administeredtopically, to the skin or mucosal tissue.

Topical formulations comprising one or more sirtuin-modulating compoundsthat increase the level and/or activity of a sirtuin protein may also beused as preventive, e.g., chemopreventive, compositions. When used in achemopreventive method, susceptible skin is treated prior to any visiblecondition in a particular individual.

Sirtuin-modulating compounds may be delivered locally or systemically toa subject. In one embodiment, a sirtuin-modulating compound is deliveredlocally to a tissue or organ of a subject by injection, topicalformulation, etc.

In another embodiment, a sirtuin-modulating compound that increases thelevel and/or activity of a sirtuin protein may be used for treating orpreventing a disease or condition induced or exacerbated by cellularsenescence in a subject; methods for decreasing the rate of senescenceof a subject, e.g., after onset of senescence; methods for extending thelifespan of a subject; methods for treating or preventing a disease orcondition relating to lifespan; methods for treating or preventing adisease or condition relating to the proliferative capacity of cells;and methods for treating or preventing a disease or condition resultingfrom cell damage or death. In certain embodiments, the method does notact by decreasing the rate of occurrence of diseases that shorten thelifespan of a subject. In certain embodiments, a method does not act byreducing the lethality caused by a disease, such as cancer.

In yet another embodiment, a sirtuin-modulating compound that increasesthe level and/or activity of a sirtuin protein may be administered to asubject in order to generally increase the lifespan of its cells and toprotect its cells against stress and/or against apoptosis. It isbelieved that treating a subject with a compound described herein issimilar to subjecting the subject to hormesis, i.e., mild stress that isbeneficial to organisms and may extend their lifespan.

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may be administered to a subject to prevent aging andaging-related consequences or diseases, such as stroke, heart disease,heart failure, arthritis, high blood pressure, and Alzheimer's disease.Other conditions that can be treated include ocular disorders, e.g.,associated with the aging of the eye, such as cataracts, glaucoma, andmacular degeneration. Sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein can also be administered tosubjects for treatment of diseases, e.g., chronic diseases, associatedwith cell death, in order to protect the cells from cell death.Exemplary diseases include those associated with neural cell death,neuronal dysfunction, or muscular cell death or dysfunction, such asParkinson's disease, Alzheimer's disease, multiple sclerosis,amniotropic lateral sclerosis, and muscular dystrophy; AIDS; fulminanthepatitis; diseases linked to degeneration of the brain, such asCreutzfeld-Jakob disease, retinitis pigmentosa and cerebellardegeneration; myelodysplasis such as aplastic anemia; ischemic diseasessuch as myocardial infarction and stroke; hepatic diseases such asalcoholic hepatitis, hepatitis B and hepatitis C; joint-diseases such asosteoarthritis; atherosclerosis; alopecia; damage to the skin due to UVlight; lichen planus; atrophy of the skin; cataract; and graftrejections. Cell death can also be caused by surgery, drug therapy,chemical exposure or radiation exposure.

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein can also be administered to a subject suffering froman acute disease, e.g., damage to an organ or tissue, e.g., a subjectsuffering from stroke or myocardial infarction or a subject sufferingfrom a spinal cord injury. Sirtuin-modulating compounds that increasethe level and/or activity of a sirtuin protein may also be used torepair an alcoholic's liver.

Cardiovascular Disease

In another embodiment, the invention provides a method for treatingand/or preventing a cardiovascular disease by administering to a subjectin need thereof a sirtuin-modulating compound that increases the leveland/or activity of a sirtuin protein.

Cardiovascular diseases that can be treated or prevented using thesirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein include cardiomyopathy or myocarditis; such asidiopathic cardiomyopathy, metabolic cardiomyopathy, alcoholiccardiomyopathy, drug-induced cardiomyopathy, ischemic cardiomyopathy,and hypertensive cardiomyopathy. Also treatable or preventable usingcompounds and methods described herein are atheromatous disorders of themajor blood vessels (macrovascular disease) such as the aorta, thecoronary arteries, the carotid arteries, the cerebrovascular arteries,the renal arteries, the iliac arteries, the femoral arteries, and thepopliteal arteries. Other vascular diseases that can be treated orprevented include those related to platelet aggregation, the retinalarterioles, the glomerular arterioles, the vasa nervorum, cardiacarterioles, and associated capillary beds of the eye, the kidney, theheart, and the central and peripheral nervous systems. Thesirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may also be used for increasing HDL levels in plasmaof an individual.

Yet other disorders that may be treated with sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteininclude restenosis, e.g., following coronary intervention, and disordersrelating to an abnormal level of high density and low densitycholesterol.

In one embodiment, a sirtuin-modulating compound that increases thelevel and/or activity of a sirtuin protein may be administered as partof a combination therapeutic with another cardiovascular agent. In oneembodiment, a sirtuin-modulating compound that increases the leveland/or activity of a sirtuin protein may be administered as part of acombination therapeutic with an anti-arrhythmia agent. In anotherembodiment, a sirtuin-modulating compound that increases the leveland/or activity of a sirtuin protein may be administered as part of acombination therapeutic with another cardiovascular agent.

Cell Death/Cancer

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may be administered to subjects who have recentlyreceived or are likely to receive a dose of radiation or toxin. In oneembodiment, the dose of radiation or toxin is received as part of awork-related or medical procedure, e.g., administered as a prophylacticmeasure. In another embodiment, the radiation or toxin exposure isreceived unintentionally. In such a case, the compound is preferablyadministered as soon as possible after the exposure to inhibit apoptosisand the subsequent development of acute radiation syndrome.

Sirtuin-modulating compounds may also be used for treating and/orpreventing cancer. In certain embodiments, sirtuin-modulating compoundsthat increase the level and/or activity of a sirtuin protein may be usedfor treating and/or preventing cancer. Calorie restriction has beenlinked to a reduction in the incidence of age-related disordersincluding cancer. Accordingly, an increase in the level and/or activityof a sirtuin protein may be useful for treating and/or preventing theincidence of age-related disorders, such as, for example, cancer.Exemplary cancers that may be treated using a sirtuin-modulatingcompound are those of the brain and kidney; hormone-dependent cancersincluding breast, prostate, testicular, and ovarian cancers; lymphomas,and leukemias. In cancers associated with solid tumors, a modulatingcompound may be administered directly into the tumor. Cancer of bloodcells, e.g., leukemia, can be treated by administering a modulatingcompound into the blood stream or into the bone marrow. Benign cellgrowth, e.g., warts, can also be treated. Other diseases that can betreated include autoimmune diseases, e.g., systemic lupus erythematosus,scleroderma, and arthritis, in which autoimmune cells should be removed.Viral infections such as herpes, HIV, adenovirus, and HTLV-1 associatedmalignant and benign disorders can also be treated by administration ofsirtuin-modulating compound. Alternatively, cells can be obtained from asubject, treated ex vivo to remove certain undesirable cells, e.g.,cancer cells, and administered back to the same or a different subject.

Chemotherapeutic agents may be co-administered with modulating compoundsdescribed herein as having anti-cancer activity, e.g., compounds thatinduce apoptosis, compounds that reduce lifespan or compounds thatrender cells sensitive to stress. Chemotherapeutic agents may be used bythemselves with a sirtuin-modulating compound described herein asinducing cell death or reducing lifespan or increasing sensitivity tostress and/or in combination with other chemotherapeutics agents. Inaddition to conventional chemotherapeutics, the sirtuin-modulatingcompounds described herein may also be used with antisense RNA, RNAi orother polynucleotides to inhibit the expression of the cellularcomponents that contribute to unwanted cellular proliferation.

Combination therapies comprising sirtuin-modulating compounds and aconventional chemotherapeutic agent may be advantageous over combinationtherapies known in the art because the combination allows theconventional chemotherapeutic agent to exert greater effect at lowerdosage. In a preferred embodiment, the effective dose (ED₅₀) for achemotherapeutic agent, or combination of conventional chemotherapeuticagents, when used in combination with a sirtuin-modulating compound isat least 2 fold less than the ED₅₀ for the chemotherapeutic agent alone,and even more preferably at 5 fold, 10 fold or even 25 fold less.Conversely, the therapeutic index (TI) for such chemotherapeutic agentor combination of such chemotherapeutic agent when used in combinationwith a sirtuin-modulating compound described herein can be at least 2fold greater than the TI for conventional chemotherapeutic regimenalone, and even more preferably at 5 fold, 10 fold or even 25 foldgreater.

Neuronal Diseases/Disorders

In certain aspects, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein can be used to treat patientssuffering from neurodegenerative diseases, and traumatic or mechanicalinjury to the central nervous system (CNS), spinal cord or peripheralnervous system (PNS). Neurodegenerative disease typically involvesreductions in the mass and volume of the human brain, which may be dueto the atrophy and/or death of brain cells, which are far more profoundthan those in a healthy person that are attributable to aging.Neurodegenerative diseases can evolve gradually, after a long period ofnormal brain function, due to progressive degeneration (e.g., nerve celldysfunction and death) of specific brain regions. Alternatively,neurodegenerative diseases can have a quick onset, such as thoseassociated with trauma or toxins. The actual onset of brain degenerationmay precede clinical expression by many years. Examples ofneurodegenerative diseases include, but are not limited to, Alzheimer'sdisease (AD), Parkinson's disease (PD), Huntington's disease (HD),amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), diffuse Lewybody disease, chorea-acanthocytosis, primary lateral sclerosis, oculardiseases (ocular neuritis), chemotherapy-induced neuropathies (e.g.,from vincristine, paclitaxel, bortezomib), diabetes-induced neuropathiesand Friedreich's ataxia. Sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein can be used to treat thesedisorders and others as described below.

AD is a CNS disorder that results in memory loss, unusual behavior,personality changes, and a decline in thinking abilities. These lossesare related to the death of specific types of brain cells and thebreakdown of connections and their supporting network (e.g. glial cells)between them. The earliest symptoms include loss of recent memory,faulty judgment, and changes in personality. PD is a CNS disorder thatresults in uncontrolled body movements, rigidity, tremor, anddyskinesia, and is associated with the death of brain cells in an areaof the brain that produces dopamine. ALS (motor neuron disease) is a CNSdisorder that attacks the motor neurons, components of the CNS thatconnect the brain to the skeletal muscles.

HD is another neurodegenerative disease that causes uncontrolledmovements, loss of intellectual faculties, and emotional disturbance.Tay-Sachs disease and Sandhoff disease are glycolipid storage diseaseswhere GM2 ganglioside and related glycolipids substrates forβ-hexosaminidase accumulate in the nervous system and trigger acuteneurodegeneration.

It is well-known that apoptosis plays a role in AIDS pathogenesis in theimmune system. However, HIV-1 also induces neurological disease, whichcan be treated with sirtuin-modulating compounds of the invention.

Neuronal loss is also a salient feature of prion diseases, such asCreutzfeldt-Jakob disease in human, BSE in cattle (mad cow disease),Scrapie Disease in sheep and goats, and feline spongiform encephalopathy(FSE) in cats. Sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be useful for treating orpreventing neuronal loss due to these prior diseases.

In another embodiment, a sirtuin-modulating compound that increases thelevel and/or activity of a sirtuin protein may be used to treat orprevent any disease or disorder involving axonopathy. Distal axonopathyis a type of peripheral neuropathy that results from some metabolic ortoxic derangement of peripheral nervous system (PNS) neurons. It is themost common response of nerves to metabolic or toxic disturbances, andas such may be caused by metabolic diseases such as diabetes, renalfailure, deficiency syndromes such as malnutrition and alcoholism, orthe effects of toxins or drugs. Those with distal axonopathies usuallypresent with symmetrical glove-stocking sensori-motor disturbances. Deeptendon reflexes and autonomic nervous system (ANS) functions are alsolost or diminished in affected areas.

Diabetic neuropathies are neuropathic disorders that are associated withdiabetes mellitus. Relatively common conditions which may be associatedwith diabetic neuropathy include third nerve palsy; mononeuropathy;mononeuritis multiplex; diabetic amyotrophy; a painful polyneuropathy;autonomic neuropathy; and thoracoabdominal neuropathy.

Peripheral neuropathy is the medical term for damage to nerves of theperipheral nervous system, which may be caused either by diseases of thenerve or from the side-effects of systemic illness. Major causes ofperipheral neuropathy include seizures, nutritional deficiencies, andHIV, though diabetes is the most likely cause.

In an exemplary embodiment, a sirtuin-modulating compound that increasesthe level and/or activity of a sirtuin protein may be used to treat orprevent multiple sclerosis (MS), including relapsing MS andmonosymptomatic MS, and other demyelinating conditions, such as, forexample, chromic inflammatory demyelinating polyneuropathy (CIDP), orsymptoms associated therewith.

In yet another embodiment, a sirtuin-modulating compound that increasesthe level and/or activity of a sirtuin protein may be used to treattrauma to the nerves, including, trauma due to disease, injury(including surgical intervention), or environmental trauma (e.g.,neurotoxins, alcoholism, etc.).

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may also be useful to prevent, treat, and alleviatesymptoms of various PNS disorders. The term “peripheral neuropathy”encompasses a wide range of disorders in which the nerves outside of thebrain and spinal cord—peripheral nerves—have been damaged. Peripheralneuropathy may also be referred to as peripheral neuritis, or if manynerves are involved, the terms polyneuropathy or polyneuritis may beused.

PNS diseases treatable with sirtuin-modulating compounds that increasethe level and/or activity of a sirtuin protein include: diabetes,leprosy, Charcot-Marie-Tooth disease, Guillain-Barré syndrome andBrachial Plexus Neuropathies (diseases of the cervical and firstthoracic roots, nerve trunks, cords, and peripheral nerve components ofthe brachial plexus.

In another embodiment, a sirtuin activating compound may be used totreat or prevent a polyglutamine disease. Exemplary polyglutaminediseases include Spinobulbar muscular atrophy (Kennedy disease),Huntington's Disease (HD), Dentatorubral-pallidoluysian atrophy (HawRiver syndrome), Spinocerebellar ataxia type 1, Spinocerebellar ataxiatype 2, Spinocerebellar ataxia type 3 (Machado-Joseph disease),Spinocerebellar ataxia type 6, Spinocerebellar ataxia type 7, andSpinocerebellar ataxia type 17.

In certain embodiments, the invention provides a method to treat acentral nervous system cell to prevent damage in response to a decreasein blood flow to the cell. Typically the severity of damage that may beprevented will depend in large part on the degree of reduction in bloodflow to the cell and the duration of the reduction. In one embodiment,apoptotic or necrotic cell death may be prevented. In still a furtherembodiment, ischemic-mediated damage, such as cytoxic edema or centralnervous system tissue anoxemia, may be prevented. In each embodiment,the central nervous system cell may be a spinal cell or a brain cell.

Another aspect encompasses administrating a sirtuin activating compoundto a subject to treat a central nervous system ischemic condition. Anumber of central nervous system ischemic conditions may be treated bythe sirtuin activating compounds described herein. In one embodiment,the ischemic condition is a stroke that results in any type of ischemiccentral nervous system damage, such as apoptotic or necrotic cell death,cytoxic edema or central nervous system tissue anoxia. The stroke mayimpact any area of the brain or be caused by any etiology commonly knownto result in the occurrence of a stroke. In one alternative of thisembodiment, the stroke is a brain stem stroke. In another alternative ofthis embodiment, the stroke is a cerebellar stroke. In still anotherembodiment, the stroke is an embolic stroke. In yet another alternative,the stroke may be a hemorrhagic stroke. In a further embodiment, thestroke is a thrombotic stroke.

In yet another aspect, a sirtuin activating compound may be administeredto reduce infarct size of the ischemic core following a central nervoussystem ischemic condition. Moreover, a sirtuin activating compound mayalso be beneficially administered to reduce the size of the ischemicpenumbra or transitional zone following a central nervous systemischemic condition.

In one embodiment, a combination drug regimen may include drugs orcompounds for the treatment or prevention of neurodegenerative disordersor secondary conditions associated with these conditions. Thus, acombination drug regimen may include one or more sirtuin activators andone or more anti-neurodegeneration agents.

Blood Coagulation Disorders

In other aspects, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein can be used to treat or preventblood coagulation disorders (or hemostatic disorders). As usedinterchangeably herein, the terms “hemostasis”, “blood coagulation,” and“blood clotting” refer to the control of bleeding, including thephysiological properties of vasoconstriction and coagulation. Bloodcoagulation assists in maintaining the integrity of mammaliancirculation after injury, inflammation, disease, congenital defect,dysfunction or other disruption. Further, the formation of blood clotsdoes not only limit bleeding in case of an injury (hemostasis), but maylead to serious organ damage and death in the context of atheroscleroticdiseases by occlusion of an important artery or vein. Thrombosis is thusblood clot formation at the wrong time and place.

Accordingly, the present invention provides anticoagulation andantithrombotic treatments aiming at inhibiting the formation of bloodclots in order to prevent or treat blood coagulation disorders, such asmyocardial infarction, stroke, loss of a limb by peripheral arterydisease or pulmonary embolism.

As used interchangeably herein, “modulating or modulation of hemostasis”and “regulating or regulation of hemostasis” includes the induction(e.g., stimulation or increase) of hemostasis, as well as the inhibition(e.g., reduction or decrease) of hemostasis.

In one aspect, the invention provides a method for reducing orinhibiting hemostasis in a subject by administering a sirtuin-modulatingcompound that increases the level and/or activity of a sirtuin protein.The compositions and methods disclosed herein are useful for thetreatment or prevention of thrombotic disorders. As used herein, theterm “thrombotic disorder” includes any disorder or conditioncharacterized by excessive or unwanted coagulation or hemostaticactivity, or a hypercoagulable state. Thrombotic disorders includediseases or disorders involving platelet adhesion and thrombusformation, and may manifest as an increased propensity to formthromboses, e.g., an increased number of thromboses, thrombosis at anearly age, a familial tendency towards thrombosis, and thrombosis atunusual sites.

In another embodiment, a combination drug regimen may include drugs orcompounds for the treatment or prevention of blood coagulation disordersor secondary conditions associated with these conditions. Thus, acombination drug regimen may include one or more sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteinand one or more anti-coagulation or anti-thrombosis agents.

Weight Control

In another aspect, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be used for treating orpreventing weight gain or obesity in a subject. For example,sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may be used, for example, to treat or preventhereditary obesity, dietary obesity, hormone related obesity, obesityrelated to the administration of medication, to reduce the weight of asubject, or to reduce or prevent weight gain in a subject. A subject inneed of such a treatment may be a subject who is obese, likely to becomeobese, overweight, or likely to become overweight. Subjects who arelikely to become obese or overweight can be identified, for example,based on family history, genetics, diet, activity level, medicationintake, or various combinations thereof.

In yet other embodiments, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be administered tosubjects suffering from a variety of other diseases and conditions thatmay be treated or prevented by promoting weight loss in the subject.Such diseases include, for example, high blood pressure, hypertension,high blood cholesterol, dyslipidemia, type 2 diabetes, insulinresistance, glucose intolerance, hyperinsulinemia, coronary heartdisease, angina pectoris, congestive heart failure, stroke, gallstones,cholescystitis and cholelithiasis, gout, osteoarthritis, obstructivesleep apnea and respiratory problems, some types of cancer (such asendometrial, breast, prostate, and colon), complications of pregnancy,poor female reproductive health (such as menstrual irregularities,infertility, irregular ovulation), bladder control problems (such asstress incontinence); uric acid nephrolithiasis; psychological disorders(such as depression, eating disorders, distorted body image, and lowself esteem). Finally, patients with AIDS can develop lipodystrophy orinsulin resistance in response to combination therapies for AIDS.

In another embodiment, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used for inhibitingadipogenesis or fat cell differentiation, whether in vitro or in vivo.Such methods may be used for treating or preventing obesity.

In other embodiments, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used for reducingappetite and/or increasing satiety, thereby causing weight loss oravoidance of weight gain. A subject in need of such a treatment may be asubject who is overweight, obese or a subject likely to becomeoverweight or obese. The method may comprise administering daily or,every other day, or once a week, a dose, e.g., in the form of a pill, toa subject. The dose may be an “appetite reducing dose.”

In an exemplary embodiment, sirtuin-modulating compounds that increasethe level and/or activity of a sirtuin protein may be administered as acombination therapy for treating or preventing weight gain or obesity.For example, one or more sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be administered incombination with one or more anti-obesity agents.

In another embodiment, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be administered to reducedrug-induced weight gain. For example, a sirtuin-modulating compoundthat increases the level and/or activity of a sirtuin protein may beadministered as a combination therapy with medications that maystimulate appetite or cause weight gain, in particular, weight gain dueto factors other than water retention.

Metabolic Disorders/Diabetes

In another aspect, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be used for treating orpreventing a metabolic disorder, such as insulin-resistance, apre-diabetic state, type II diabetes, and/or complications thereof.Administration of a sirtuin-modulating compounds that increases thelevel and/or activity of a sirtuin protein may increase insulinsensitivity and/or decrease insulin levels in a subject. A subject inneed of such a treatment may be a subject who has insulin resistance orother precursor symptom of type II diabetes, who has type II diabetes,or who is likely to develop any of these conditions. For example, thesubject may be a subject having insulin resistance, e.g., having highcirculating levels of insulin and/or associated conditions, such ashyperlipidemia, dyslipogenesis, hypercholesterolemia, impaired glucosetolerance, high blood glucose sugar level, other manifestations ofsyndrome X, hypertension, atherosclerosis and lipodystrophy.

In an exemplary embodiment, sirtuin-modulating compounds that increasethe level and/or activity of a sirtuin protein may be administered as acombination therapy for treating or preventing a metabolic disorder. Forexample, one or more sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be administered incombination with one or more anti-diabetic agents.

Inflammatory Diseases

In other aspects, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein can be used to treat or prevent adisease or disorder associated with inflammation. Sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteinmay be administered prior to the onset of, at, or after the initiationof inflammation. When used prophylactically, the compounds arepreferably provided in advance of any inflammatory response or symptom.Administration of the compounds may prevent or attenuate inflammatoryresponses or symptoms.

In another embodiment, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used to treat orprevent allergies and respiratory conditions, including asthma,bronchitis, pulmonary fibrosis, allergic rhinitis, oxygen toxicity,emphysema, chronic bronchitis, acute respiratory distress syndrome, andany chronic obstructive pulmonary disease (COPD). The compounds may beused to treat chronic hepatitis infection, including hepatitis B andhepatitis C.

Additionally, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be used to treat autoimmunediseases and/or inflammation associated with autoimmune diseases such asorgan-tissue autoimmune diseases (e.g., Raynaud's syndrome),scleroderma, myasthenia gravis, transplant rejection, endotoxin shock,sepsis, psoriasis, eczema, dermatitis, multiple sclerosis, autoimmunethyroiditis, uveitis, systemic lupus erythematosis, Addison's disease,autoimmune polyglandular disease (also known as autoimmune polyglandularsyndrome), and Grave's disease.

In certain embodiments, one or more sirtuin-modulating compounds thatincrease the level and/or activity of a sirtuin protein may be takenalone or in combination with other compounds useful for treating orpreventing inflammation.

Flushing

In another aspect, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be used for reducing theincidence or severity of flushing and/or hot flashes which are symptomsof a disorder. For instance, the subject method includes the use ofsirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein, alone or in combination with other agents, forreducing incidence or severity of flushing and/or hot flashes in cancerpatients. In other embodiments, the method provides for the use ofsirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein to reduce the incidence or severity of flushing and/orhot flashes in menopausal and post-menopausal woman.

In another aspect, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be used as a therapy forreducing the incidence or severity of flushing and/or hot flashes whichare side-effects of another drug therapy, e.g., drug-induced flushing.In certain embodiments, a method for treating and/or preventingdrug-induced flushing comprises administering to a patient in needthereof a formulation comprising at least one flushing inducing compoundand at least one sirtuin-modulating compound that increases the leveland/or activity of a sirtuin protein. In other embodiments, a method fortreating drug induced flushing comprises separately administering one ormore compounds that induce flushing and one or more sirtuin-modulatingcompounds, e.g., wherein the sirtuin-modulating compound and flushinginducing agent have not been formulated in the same compositions. Whenusing separate formulations, the sirtuin-modulating compound may beadministered (1) at the same as administration of the flushing inducingagent, (2) intermittently with the flushing inducing agent, (3)staggered relative to administration of the flushing inducing agent, (4)prior to administration of the flushing inducing agent, (5) subsequentto administration of the flushing inducing agent, and (6) variouscombination thereof. Exemplary flushing inducing agents include, forexample, niacin, faloxifene, antidepressants, anti-psychotics,chemotherapeutics, calcium channel blockers, and antibiotics.

In one embodiment, sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may be used to reduce flushing sideeffects of a vasodilator or an antilipemic agent (includinganticholesteremic agents and lipotropic agents). In an exemplaryembodiment, a sirtuin-modulating compound that increases the leveland/or activity of a sirtuin protein may be used to reduce flushingassociated with the administration of niacin.

In another embodiment, the invention provides a method for treatingand/or preventing hyperlipidemia with reduced flushing side effects. Inanother representative embodiment, the method involves the use ofsirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein to reduce flushing side effects of raloxifene. Inanother representative embodiment, the method involves the use ofsirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein to reduce flushing side effects of antidepressants oranti-psychotic agent. For instance, sirtuin-modulating compounds thatincrease the level and/or activity of a sirtuin protein can be used inconjunction (administered separately or together) with a serotoninreuptake inhibitor, or a 5HT2 receptor antagonist.

In certain embodiments, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used as part of atreatment with a serotonin reuptake inhibitor (SRI) to reduce flushing.In still another representative embodiment, sirtuin-modulating compoundsthat increase the level and/or activity of a sirtuin protein may be usedto reduce flushing side effects of chemotherapeutic agents, such ascyclophosphamide and tamoxifen.

In another embodiment, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used to reduceflushing side effects of calcium channel blockers, such as amlodipine.

In another embodiment, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used to reduceflushing side effects of antibiotics. For example, sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteincan be used in combination with levofloxacin.

Ocular Disorders

One aspect of the present invention is a method for inhibiting, reducingor otherwise treating vision impairment by administering to a patient atherapeutic dosage of sirtuin modulator selected from a compounddisclosed herein, or a pharmaceutically acceptable salt, prodrug or ametabolic derivative thereof.

In certain aspects of the invention, the vision impairment is caused bydamage to the optic nerve or central nervous system. In particularembodiments, optic nerve damage is caused by high intraocular pressure,such as that created by glaucoma. In other particular embodiments, opticnerve damage is caused by swelling of the nerve, which is oftenassociated with an infection or an immune (e.g., autoimmune) responsesuch as in optic neuritis.

In certain aspects of the invention, the vision impairment is caused byretinal damage. In particular embodiments, retinal damage is caused bydisturbances in blood flow to the eye (e.g., arteriosclerosis,vasculitis). In particular embodiments, retinal damage is caused bydisruption of the macula (e.g., exudative or non-exudative maculardegeneration).

Exemplary retinal diseases include Exudative Age Related MacularDegeneration, Nonexudative Age Related Macular Degeneration, RetinalElectronic Prosthesis and RPE Transplantation Age Related MacularDegeneration, Acute Multifocal Placoid Pigment Epitheliopathy, AcuteRetinal Necrosis, Best Disease, Branch Retinal Artery Occlusion, BranchRetinal Vein Occlusion, Cancer Associated and Related AutoimmuneRetinopathies, Central Retinal Artery Occlusion, Central Retinal VeinOcclusion, Central Serous Chorioretinopathy, Eales Disease, EpimacularMembrane, Lattice Degeneration, Macroaneurysm, Diabetic Macular Edema,Irvine-Gass Macular Edema, Macular Hole, Subretinal NeovascularMembranes, Diffuse Unilateral Subacute Neuroretinitis, NonpseudophakicCystoid Macular Edema, Presumed Ocular Histoplasmosis Syndrome,Exudative Retinal Detachment, Postoperative Retinal Detachment,Proliferative Retinal Detachment, Rhegmatogenous Retinal Detachment,Tractional Retinal Detachment, Retinitis Pigmentosa, CMV Retinitis,Retinoblastoma, Retinopathy of Prematurity, Birdshot Retinopathy,Background Diabetic Retinopathy, Proliferative Diabetic Retinopathy,Hemoglobinopathies Retinopathy, Purtscher Retinopathy, ValsalvaRetinopathy, Juvenile Retinoschisis, Senile Retinoschisis, TersonSyndrome and White Dot Syndromes.

Other exemplary diseases include ocular bacterial infections (e.g.conjunctivitis, keratitis, tuberculosis, syphilis, gonorrhea), viralinfections (e.g. Ocular Herpes Simplex Virus, Varicella Zoster Virus,Cytomegalovirus retinitis, Human Immunodeficiency Virus (HIV)) as wellas progressive outer retinal necrosis secondary to HIV or otherHIV-associated and other immunodeficiency-associated ocular diseases. Inaddition, ocular diseases include fungal infections (e.g. Candidachoroiditis, histoplasmosis), protozoal infections (e.g. toxoplasmosis)and others such as ocular toxocariasis and sarcoidosis.

One aspect of the invention is a method for inhibiting, reducing ortreating vision impairment in a subject undergoing treatment with achemotherapeutic drug (e.g., a neurotoxic drug, a drug that raisesintraocular pressure such as a steroid), by administering to the subjectin need of such treatment a therapeutic dosage of a sirtuin modulatordisclosed herein.

Another aspect of the invention is a method for inhibiting, reducing ortreating vision impairment in a subject undergoing surgery, includingocular or other surgeries performed in the prone position such as spinalcord surgery, by administering to the subject in need of such treatmenta therapeutic dosage of a sirtuin modulator disclosed herein. Ocularsurgeries include cataract, iridotomy and lens replacements.

Another aspect of the invention is the treatment, including inhibitionand prophylactic treatment, of age related ocular diseases includecataracts, dry eye, age-related macular degeneration (AMD), retinaldamage and the like, by administering to the subject in need of suchtreatment a therapeutic dosage of a sirtuin modulator disclosed herein.

Another aspect of the invention is the prevention or treatment of damageto the eye caused by stress, chemical insult or radiation, byadministering to the subject in need of such treatment a therapeuticdosage of a sirtuin modulator disclosed herein. Radiation orelectromagnetic damage to the eye can include that caused by CRT's orexposure to sunlight or UV.

In one embodiment, a combination drug regimen may include drugs orcompounds for the treatment or prevention of ocular disorders orsecondary conditions associated with these conditions. Thus, acombination drug regimen may include one or more sirtuin activators andone or more therapeutic agents for the treatment of an ocular disorder.

In one embodiment, a sirtuin modulator can be administered inconjunction with a therapy for reducing intraocular pressure. In anotherembodiment, a sirtuin modulator can be administered in conjunction witha therapy for treating and/or preventing glaucoma. In yet anotherembodiment, a sirtuin modulator can be administered in conjunction witha therapy for treating and/or preventing optic neuritis. In oneembodiment, a sirtuin modulator can be administered in conjunction witha therapy for treating and/or preventing CMV Retinopathy. In anotherembodiment, a sirtuin modulator can be administered in conjunction witha therapy for treating and/or preventing multiple sclerosis.

Mitochondrial-Associated Diseases and Disorders

In certain embodiments, the invention provides methods for treatingdiseases or disorders that would benefit from increased mitochondrialactivity. The methods involve administering to a subject in need thereofa therapeutically effective amount of a sirtuin activating compound.Increased mitochondrial activity refers to increasing activity of themitochondria while maintaining the overall numbers of mitochondria(e.g., mitochondrial mass), increasing the numbers of mitochondriathereby increasing mitochondrial activity (e.g., by stimulatingmitochondrial biogenesis), or combinations thereof. In certainembodiments, diseases and disorders that would benefit from increasedmitochondrial activity include diseases or disorders associated withmitochondrial dysfunction.

In certain embodiments, methods for treating diseases or disorders thatwould benefit from increased mitochondrial activity may compriseidentifying a subject suffering from a mitochondrial dysfunction.Methods for diagnosing a mitochondrial dysfunction may involve moleculargenetic, pathologic and/or biochemical analyses. Diseases and disordersassociated with mitochondrial dysfunction include diseases and disordersin which deficits in mitochondrial respiratory chain activity contributeto the development of pathophysiology of such diseases or disorders in amammal. Diseases or disorders that would benefit from increasedmitochondrial activity generally include for example, diseases in whichfree radical mediated oxidative injury leads to tissue degeneration,diseases in which cells inappropriately undergo apoptosis, and diseasesin which cells fail to undergo apoptosis.

In certain embodiments, the invention provides methods for treating adisease or disorder that would benefit from increased mitochondrialactivity that involves administering to a subject in need thereof one ormore sirtuin activating compounds in combination with anothertherapeutic agent such as, for example, an agent useful for treatingmitochondrial dysfunction or an agent useful for reducing a symptomassociated with a disease or disorder involving mitochondrialdysfunction.

In exemplary embodiments, the invention provides methods for treatingdiseases or disorders that would benefit from increased mitochondrialactivity by administering to a subject a therapeutically effectiveamount of a sirtuin activating compound. Exemplary diseases or disordersinclude, for example, neuromuscular disorders (e.g., Friedreich'sAtaxia, muscular dystrophy, multiple sclerosis, etc.), disorders ofneuronal instability (e.g., seizure disorders, migrane, etc.),developmental delay, neurodegenerative disorders (e.g., Alzheimer'sDisease, Parkinson's Disease, amyotrophic lateral sclerosis, etc.),ischemia, renal tubular acidosis, age-related neurodegeneration andcognitive decline, chemotherapy fatigue, age-related orchemotherapy-induced menopause or irregularities of menstrual cycling orovulation, mitochondrial myopathies, mitochondrial damage (e.g., calciumaccumulation, excitotoxicity, nitric oxide exposure, hypoxia, etc.), andmitochondrial deregulation.

Muscular dystrophy refers to a family of diseases involvingdeterioration of neuromuscular structure and function, often resultingin atrophy of skeletal muscle and myocardial dysfunction, such asDuchenne muscular dystrophy. In certain embodiments, sirtuin activatingcompounds may be used for reducing the rate of decline in muscularfunctional capacities and for improving muscular functional status inpatients with muscular dystrophy.

In certain embodiments, sirtuin modulating compounds may be useful fortreatment mitochondrial myopathies. Mitochondrial myopathies range frommild, slowly progressive weakness of the extraocular muscles to severe,fatal infantile myopathies and multisystem encephalomyopathies. Somesyndromes have been defined, with some overlap between them. Establishedsyndromes affecting muscle include progressive external opthalmoplegia,the Kearns-Sayre syndrome (with opthalmoplegia, pigmentary retinopathy,cardiac conduction defects, cerebellar ataxia, and sensorineuraldeafness), the MELAS syndrome (mitochondrial encephalomyopathy, lacticacidosis, and stroke-like episodes), the MERFF syndrome (myoclonicepilepsy and ragged red fibers), limb-girdle distribution weakness, andinfantile myopathy (benign or severe and fatal).

In certain embodiments, sirtuin activating compounds may be useful fortreating patients suffering from toxic damage to mitochondria, such as,toxic damage due to calcium accumulation, excitotoxicity, nitric oxideexposure, drug induced toxic damage, or hypoxia.

In certain embodiments, sirtuin activating compounds may be useful fortreating diseases or disorders associated with mitochondrialderegulation.

Muscle Performance

In other embodiments, the invention provides methods for enhancingmuscle performance by administering a therapeutically effective amountof a sirtuin activating compound. For example, sirtuin activatingcompounds may be useful for improving physical endurance (e.g., abilityto perform a physical task such as exercise, physical labor, sportsactivities, etc.), inhibiting or retarding physical fatigues, enhancingblood oxygen levels, enhancing energy in healthy individuals, enhanceworking capacity and endurance, reducing muscle fatigue, reducingstress, enhancing cardiac and cardiovascular function, improving sexualability, increasing muscle ATP levels, and/or reducing lactic acid inblood. In certain embodiments, the methods involve administering anamount of a sirtuin activating compound that increase mitochondrialactivity, increase mitochondrial biogenesis, and/or increasemitochondrial mass.

Sports performance refers to the ability of the athlete's muscles toperform when participating in sports activities. Enhanced sportsperformance, strength, speed and endurance are measured by an increasein muscular contraction strength, increase in amplitude of musclecontraction, shortening of muscle reaction time between stimulation andcontraction. Athlete refers to an individual who participates in sportsat any level and who seeks to achieve an improved level of strength,speed and endurance in their performance, such as, for example, bodybuilders, bicyclists, long distance runners, short distance runners,etc. Enhanced sports performance in manifested by the ability toovercome muscle fatigue, ability to maintain activity for longer periodsof time, and have a more effective workout.

In the arena of athlete muscle performance, it is desirable to createconditions that permit competition or training at higher levels ofresistance for a prolonged period of time.

It is contemplated that the methods of the present invention will alsobe effective in the treatment of muscle related pathological conditions,including acute sarcopenia, for example, muscle atrophy and/or cachexiaassociated with burns, bed rest, limb immobilization, or major thoracic,abdominal, and/or orthopedic surgery.

In certain embodiments, the invention provides novel dietarycompositions comprising sirtuin modulators, a method for theirpreparation, and a method of using the compositions for improvement ofsports performance. Accordingly, provided are therapeutic compositions,foods and beverages that have actions of improving physical enduranceand/or inhibiting physical fatigues for those people involved inbroadly-defined exercises including sports requiring endurance andlabors requiring repeated muscle exertions. Such dietary compositionsmay additional comprise electrolytes, caffeine, vitamins, carbohydrates,etc.

Other Uses

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may be used for treating or preventing viralinfections (such as infections by influenza, herpes or papilloma virus)or as antifungal agents. In certain embodiments, sirtuin-modulatingcompounds that increase the level and/or activity of a sirtuin proteinmay be administered as part of a combination drug therapy with anothertherapeutic agent for the treatment of viral diseases. In anotherembodiment, sirtuin-modulating compounds that increase the level and/oractivity of a sirtuin protein may be administered as part of acombination drug therapy with another anti-fungal agent.

Subjects that may be treated as described herein include eukaryotes,such as mammals, e.g., humans, ovines, bovines, equines, porcines,canines, felines, non-human primate, mice, and rats. Cells that may betreated include eukaryotic cells, e.g., from a subject described above,or plant cells, yeast cells and prokaryotic cells, e.g., bacterialcells. For example, modulating compounds may be administered to farmanimals to improve their ability to withstand farming conditions longer.

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may also be used to increase lifespan, stressresistance, and resistance to apoptosis in plants. In one embodiment, acompound is applied to plants, e.g., on a periodic basis, or to fungi.In another embodiment, plants are genetically modified to produce acompound. In another embodiment, plants and fruits are treated with acompound prior to picking and shipping to increase resistance to damageduring shipping. Plant seeds may also be contacted with compoundsdescribed herein, e.g., to preserve them.

In other embodiments, sirtuin-modulating compounds that increase thelevel and/or activity of a sirtuin protein may be used for modulatinglifespan in yeast cells. Situations in which it may be desirable toextend the lifespan of yeast cells include any process in which yeast isused, e.g., the making of beer, yogurt, and bakery items, e.g., bread.Use of yeast having an extended lifespan can result in using less yeastor in having the yeast be active for longer periods of time. Yeast orother mammalian cells used for recombinantly producing proteins may alsobe treated as described herein.

Sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein may also be used to increase lifespan, stressresistance and resistance to apoptosis in insects. In this embodiment,compounds would be applied to useful insects, e.g., bees and otherinsects that are involved in pollination of plants. In a specificembodiment, a compound would be applied to bees involved in theproduction of honey. Generally, the methods described herein may beapplied to any organism, e.g., eukaryote, that may have commercialimportance. For example, they can be applied to fish (aquaculture) andbirds (e.g., chicken and fowl).

Higher doses of sirtuin-modulating compounds that increase the leveland/or activity of a sirtuin protein may also be used as a pesticide byinterfering with the regulation of silenced genes and the regulation ofapoptosis during development. In this embodiment, a compound may beapplied to plants using a method known in the art that ensures thecompound is bio-available to insect larvae, and not to plants.

At least in view of the link between reproduction and longevity,sirtuin-modulating compounds that increase the level and/or activity ofa sirtuin protein can be applied to affect the reproduction of organismssuch as insects, animals and microorganisms.

4. Assays

Various types of assays to determine sirtuin activity have beendescribed. For example, sirtuin activity may be determined using afluorescence based assay such as the assay commercially available fromBiomol, e.g., the SIRT1 Fluorimetric Drug Discovery Kit (AK-555), SIRT2Fluorimetric Drug Discovery Kit (AK-556), or SIRT3 Fluorimetric DrugDiscovery Kit (AK-557) (Biomol International, Plymouth Meeting, Pa.).Other suitable sirtuin assays include a nicotinamide release assay(Kaeberlein et al., J. Biol. Chem. 280(17): 17038 (2005)), a FRET assay(Marcotte et al., Anal. Biochem. 332: 90 (2004)), and a C¹⁴ NAD boronresin binding assay (McDonagh et al., Methods 36: 346 (2005)). Yet othersuitable sirtuin assays include radioimmunoassays (RIA), scintillationproximity assays, HPLC based assays, and reporter gene assays (e.g., fortranscription factor targets).

An exemplary assay for determining sirtuin activity is a fluorescencepolarization assay. Fluorescence polarization assays are describedherein and are also described in PCT Publication No. WO 2006/094239. Inother embodiments, sirtuin activity may be determined using a massspectrometry based assays. Examples of mass spectrometry based assaysare described herein and are also described in PCT Publication No. WO2007/064902. Cell based assays may also be used to determine sirtuinactivity. Examples of cell based assays for determining sirtuin activityare described in PCT Publication Nos. WO 2007/064902 and WO 2008/060400.

Yet other methods contemplated herein include screening methods foridentifying compounds or agents that modulate sirtuins. An agent may bea nucleic acid, such as an aptamer. Assays may be conducted in a cellbased or cell free format. For example, an assay may comprise incubating(or contacting) a sirtuin with a test agent under conditions in which asirtuin can be modulated by an agent known to modulate the sirtuin, andmonitoring or determining the level of modulation of the sirtuin in thepresence of the test agent relative to the absence of the test agent.The level of modulation of a sirtuin can be determined by determiningits ability to deacetylate a substrate. Exemplary substrates areacetylated peptides which can be obtained from BIOMOL (Plymouth Meeting,Pa.). Preferred substrates include peptides of p53, such as thosecomprising an acetylated K382. A particularly preferred substrate is theFluor de Lys-SIRT1 (BIOMOL), i.e., the acetylated peptideArg-His-Lys-Lys. Other substrates are peptides from human histones H3and H4 or an acetylated amino acid. Substrates may be fluorogenic. Thesirtuin may be SIRT1, Sir2, SIRT3, or a portion thereof. For example,recombinant SIRT1 can be obtained from BIOMOL. The reaction may beconducted for about 30 minutes and stopped, e.g., with nicotinamide. TheHDAC fluorescent activity assay/drug discovery kit (AK-500, BIOMOLResearch Laboratories) may be used to determine the level ofacetylation. Similar assays are described in Bitterman et al. (2002) J.Biol. Chem. 277:45099. The level of modulation of the sirtuin in anassay may be compared to the level of modulation of the sirtuin in thepresence of one or more (separately or simultaneously) compoundsdescribed herein, which may serve as positive or negative controls.Sirtuins for use in the assays may be full length sirtuin proteins orportions thereof. Since it has been shown herein that activatingcompounds appear to interact with the N-terminus of SIRT1, proteins foruse in the assays include N-terminal portions of sirtuins, e.g., aboutamino acids 1-176 or 1-255 of SIRT1; about amino acids 1-174 or 1-252 ofSir2.

In one embodiment, a screening assay comprises (i) contacting a sirtuinwith a test agent and an acetylated substrate under conditionsappropriate for the sirtuin to deacetylate the substrate in the absenceof the test agent; and (ii) determining the level of acetylation of thesubstrate, wherein a lower level of acetylation of the substrate in thepresence of the test agent relative to the absence of the test agentindicates that the test agent stimulates deacetylation by the sirtuin,whereas a higher level of acetylation of the substrate in the presenceof the test agent relative to the absence of the test agent indicatesthat the test agent inhibits deacetylation by the sirtuin.

Methods for identifying an agent that modulates, e.g., stimulates,sirtuins in vivo may comprise (i) contacting a cell with a test agentand a substrate that is capable of entering a cell in the presence of aninhibitor of class I and class II HDACs under conditions appropriate forthe sirtuin to deacetylate the substrate in the absence of the testagent; and (ii) determining the level of acetylation of the substrate,wherein a lower level of acetylation of the substrate in the presence ofthe test agent relative to the absence of the test agent indicates thatthe test agent stimulates deacetylation by the sirtuin, whereas a higherlevel of acetylation of the substrate in the presence of the test agentrelative to the absence of the test agent indicates that the test agentinhibits deacetylation by the sirtuin. A preferred substrate is anacetylated peptide, which is also preferably fluorogenic, as furtherdescribed herein. The method may further comprise lysing the cells todetermine the level of acetylation of the substrate. Substrates may beadded to cells at a concentration ranging from about 1 μM to about 10mM, preferably from about 10 μM to 1 mM, even more preferably from about100 μM to 1 mM, such as about 200 μM. A preferred substrate is anacetylated lysine, e.g., ε-acetyl lysine (Fluor de Lys, FdL) or Fluor deLys-SIRT1. A preferred inhibitor of class I and class II HDACs istrichostatin A (TSA), which may be used at concentrations ranging fromabout 0.01 to 100 μM, preferably from about 0.1 to 10 μM, such as 1 μM.Incubation of cells with the test compound and the substrate may beconducted for about 10 minutes to 5 hours, preferably for about 1-3hours. Since TSA inhibits all class I and class II HDACs, and thatcertain substrates, e.g., Fluor de Lys, is a poor substrate for SIRT2and even less a substrate for SIRT3-7, such an assay may be used toidentify modulators of SIRT1 in vivo.

5. Pharmaceutical Compositions

The sirtuin-modulating compounds described herein may be formulated in aconventional manner using one or more physiologically acceptablecarriers or excipients. For example, sirtuin-modulating compounds andtheir physiologically acceptable salts and solvates may be formulatedfor administration by, for example, injection (e.g. SubQ, IM, IP),inhalation or insufflation (either through the mouth or the nose) ororal, buccal, sublingual, transdermal, nasal, parenteral or rectaladministration. In one embodiment, a sirtuin-modulating compound may beadministered locally, at the site where the target cells are present,i.e., in a specific tissue, organ, or fluid (e.g., blood, cerebrospinalfluid, etc.).

Sirtuin-modulating compounds can be formulated for a variety of modes ofadministration, including systemic and topical or localizedadministration. Techniques and formulations generally may be found inRemington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa.For parenteral administration, injection is preferred, includingintramuscular, intravenous, intraperitoneal, and subcutaneous. Forinjection, the compounds can be formulated in liquid solutions,preferably in physiologically compatible buffers such as Hank's solutionor Ringer's solution. In addition, the compounds may be formulated insolid form and redissolved or suspended immediately prior to use.Lyophilized forms are also included.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets, lozenges, or capsules prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g., magnesium stearate, talc or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulphate). The tablets may be coated by methods well known in theart. Liquid preparations for oral administration may take the form of,for example, solutions, syrups or suspensions, or they may be presentedas a dry product for constitution with water or other suitable vehiclebefore use. Such liquid preparations may be prepared by conventionalmeans with pharmaceutically acceptable additives such as suspendingagents (e.g., sorbitol syrup, cellulose derivatives or hydrogenatededible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueousvehicles (e.g., ationd oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations may alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate. Preparations for oral administration may be suitablyformulated to give controlled release of the active compound.

For administration by inhalation (e.g., pulmonary delivery),sirtuin-modulating compounds may be conveniently delivered in the formof an aerosol spray presentation from pressurized packs or a nebuliser,with the use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin, for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

Sirtuin-modulating compounds may be formulated for parenteraladministration by injection, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

Sirtuin-modulating compounds may also be formulated in rectalcompositions such as suppositories or retention enemas, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, sirtuin-modulatingcompounds may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, sirtuin-modulating compounds may be formulated withsuitable polymeric or hydrophobic materials (for example as an emulsionin an acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt. Controlledrelease formula also includes patches.

In certain embodiments, the compounds described herein can be formulatedfor delivery to the central nervous system (CNS) (reviewed in Begley,Pharmacology & Therapeutics 104: 29-45 (2004)). Conventional approachesfor drug delivery to the CNS include: neurosurgical strategies (e.g.,intracerebral injection or intracerebroventricular infusion); molecularmanipulation of the agent (e.g., production of a chimeric fusion proteinthat comprises a transport peptide that has an affinity for anendothelial cell surface molecule in combination with an agent that isitself incapable of crossing the BBB) in an attempt to exploit one ofthe endogenous transport pathways of the BBB; pharmacological strategiesdesigned to increase the lipid solubility of an agent (e.g., conjugationof water-soluble agents to lipid or cholesterol carriers); and thetransitory disruption of the integrity of the BBB by hyperosmoticdisruption (resulting from the infusion of a mannitol solution into thecarotid artery or the use of a biologically active agent such as anangiotensin peptide).

Liposomes are a further drug delivery system which is easily injectable.Accordingly, in the method of invention the active compounds can also beadministered in the form of a liposome delivery system. Liposomes arewell-known by a person skilled in the art. Liposomes can be formed froma variety of phospholipids, such as cholesterol, stearylamine ofphosphatidylcholines. Liposomes being usable for the method of inventionencompass all types of liposomes including, but not limited to, smallunilamellar vesicles, large unilamellar vesicles and multilamellarvesicles.

Another way to produce a formulation, particularly a solution, of asirtuin modulator such as resveratrol or a derivative thereof, isthrough the use of cyclodextrin. By cyclodextrin is meant α-, β-, orγ-cyclodextrin. Cyclodextrins are described in detail in Pitha et al.,U.S. Pat. No. 4,727,064, which is incorporated herein by reference.Cyclodextrins are cyclic oligomers of glucose; these compounds forminclusion complexes with any drug whose molecule can fit into thelipophile-seeking cavities of the cyclodextrin molecule.

Rapidly disintegrating or dissolving dosage forms are useful for therapid absorption, particularly buccal and sublingual absorption, ofpharmaceutically active agents. Fast melt dosage forms are beneficial topatients, such as aged and pediatric patients, who have difficulty inswallowing typical solid dosage forms, such as caplets and tablets.Additionally, fast melt dosage forms circumvent drawbacks associatedwith, for example, chewable dosage forms, wherein the length of time anactive agent remains in a patient's mouth plays an important role indetermining the amount of taste masking and the extent to which apatient may object to throat grittiness of the active agent.

Pharmaceutical compositions (including cosmetic preparations) maycomprise from about 0.00001 to 100% such as from 0.001 to 10% or from0.1% to 5% by weight of one or more sirtuin-modulating compoundsdescribed herein.

In one embodiment, a sirtuin-modulating compound described herein, isincorporated into a topical formulation containing a topical carrierthat is generally suited to topical drug administration and comprisingany such material known in the art. The topical carrier may be selectedso as to provide the composition in the desired form, e.g., as anointment, lotion, cream, microemulsion, gel, oil, solution, or the like,and may be comprised of a material of either naturally occurring orsynthetic origin. It is preferable that the selected carrier notadversely affect the active agent or other components of the topicalformulation. Examples of suitable topical carriers for use hereininclude water, alcohols and other nontoxic organic solvents, glycerin,mineral oil, silicone, petroleum jelly, lanolin, fatty acids, vegetableoils, parabens, waxes, and the like.

Formulations may be colorless, odorless ointments, lotions, creams,microemulsions and gels.

Sirtuin-modulating compounds may be incorporated into ointments, whichgenerally are semisolid preparations which are typically based onpetrolatum or other petroleum derivatives. The specific ointment base tobe used, as will be appreciated by those skilled in the art, is one thatwill provide for optimum drug delivery, and, preferably, will providefor other desired characteristics as well, e.g., emolliency or the like.As with other carriers or vehicles, an ointment base should be inert,stable, nonirritating and nonsensitizing.

Sirtuin-modulating compounds may be incorporated into lotions, whichgenerally are preparations to be applied to the skin surface withoutfriction, and are typically liquid or semiliquid preparations in whichsolid particles, including the active agent, are present in a water oralcohol base. Lotions are usually suspensions of solids, and maycomprise a liquid oily emulsion of the oil-in-water type.

Sirtuin-modulating compounds may be incorporated into creams, whichgenerally are viscous liquid or semisolid emulsions, either oil-in-wateror water-in-oil. Cream bases are water-washable, and contain an oilphase, an emulsifier and an aqueous phase. The oil phase is generallycomprised of petrolatum and a fatty alcohol such as cetyl or stearylalcohol; the aqueous phase usually, although not necessarily, exceedsthe oil phase in volume, and generally contains a humectant. Theemulsifier in a cream formulation, as explained in Remington's, supra,is generally a nonionic, anionic, cationic or amphoteric surfactant.

Sirtuin-modulating compounds may be incorporated into microemulsions,which generally are thermodynamically stable, isotropically cleardispersions of two immiscible liquids, such as oil and water, stabilizedby an interfacial film of surfactant molecules (Encyclopedia ofPharmaceutical Technology (New York: Marcel Dekker, 1992), volume 9).

Sirtuin-modulating compounds may be incorporated into gel formulations,which generally are semisolid systems consisting of either suspensionsmade up of small inorganic particles (two-phase systems) or largeorganic molecules distributed substantially uniformly throughout acarrier liquid (single phase gels). Although gels commonly employaqueous carrier liquid, alcohols and oils can be used as the carrierliquid as well.

Other active agents may also be included in formulations, e.g., otheranti-inflammatory agents, analgesics, antimicrobial agents, antifungalagents, antibiotics, vitamins, antioxidants, and sunblock agentscommonly found in sunscreen formulations including, but not limited to,anthranilates, benzophenones (particularly benzophenone-3), camphorderivatives, cinnamates (e.g., octyl methoxycinnamate), dibenzoylmethanes (e.g., butyl methoxydibenzoyl methane), p-aminobenzoic acid(PABA) and derivatives thereof, and salicylates (e.g., octylsalicylate).

In certain topical formulations, the active agent is present in anamount in the range of approximately 0.25 wt. % to 75 wt. % of theformulation, preferably in the range of approximately 0.25 wt. % to 30wt. % of the formulation, more preferably in the range of approximately0.5 wt. % to 15 wt. % of the formulation, and most preferably in therange of approximately 1.0 wt. % to 10 wt. % of the formulation.

Conditions of the eye can be treated or prevented by, e.g., systemic,topical, intraocular injection of a sirtuin-modulating compound, or byinsertion of a sustained release device that releases asirtuin-modulating compound. A sirtuin-modulating compound thatincreases the level and/or activity of a sirtuin protein may bedelivered in a pharmaceutically acceptable ophthalmic vehicle, such thatthe compound is maintained in contact with the ocular surface for asufficient time period to allow the compound to penetrate the cornealand internal regions of the eye, as for example the anterior chamber,posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea,iris/ciliary, lens, choroid/retina and sclera. Thepharmaceutically-acceptable ophthalmic vehicle may, for example, be anointment, vegetable oil or an encapsulating material. Alternatively, thecompounds of the invention may be injected directly into the vitreousand aqueous humour. In a further alternative, the compounds may beadministered systemically, such as by intravenous infusion or injection,for treatment of the eye.

Sirtuin-modulating compounds described herein may be stored in oxygenfree environment. For example, resveratrol or analog thereof can beprepared in an airtight capsule for oral administration, such asCapsugel from Pfizer, Inc.

Cells, e.g., treated ex vivo with a sirtuin-modulating compound, can beadministered according to methods for administering a graft to asubject, which may be accompanied, e.g., by administration of animmunosuppressant drug, e.g., cyclosporin A. For general principles inmedicinal formulation, the reader is referred to Cell Therapy: Stem CellTransplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn& W. Sheridan eds, Cambridge University Press, 1996; and HematopoieticStem Cell Therapy, E. D. Ball, J. Lister & P. Law, ChurchillLivingstone, 2000.

Toxicity and therapeutic efficacy of sirtuin-modulating compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals. The LD₅₀ is the dose lethal to 50% of thepopulation. The ED₅₀ is the dose therapeutically effective in 50% of thepopulation. The dose ratio between toxic and therapeutic effects(LD₅₀/ED₅₀) is the therapeutic index. Sirtuin-modulating compounds thatexhibit large therapeutic indexes are preferred. Whilesirtuin-modulating compounds that exhibit toxic side effects may beused, care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds may lie within a range of circulating concentrations thatinclude the ED₅₀ with little or no toxicity. The dosage may vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. For any compound, the therapeutically effectivedose can be estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

6. Kits

Also provided herein are kits, e.g., kits for therapeutic purposes orkits for modulating the lifespan of cells or modulating apoptosis. A kitmay comprise one or more sirtuin-modulating compounds, e.g., inpremeasured doses. A kit may optionally comprise devices for contactingcells with the compounds and instructions for use. Devices includesyringes, stents and other devices for introducing a sirtuin-modulatingcompound into a subject (e.g., the blood vessel of a subject) orapplying it to the skin of a subject.

In yet another embodiment, the invention provides a composition ofmatter comprising a sirtuin modulator of this invention and anothertherapeutic agent (the same ones used in combination therapies andcombination compositions) in separate dosage forms, but associated withone another. The term “associated with one another” as used herein meansthat the separate dosage forms are packaged together or otherwiseattached to one another such that it is readily apparent that theseparate dosage forms are intended to be sold and administered as partof the same regimen. The agent and the sirtuin modulator are preferablypackaged together in a blister pack or other multi-chamber package, oras connected, separately sealed containers (such as foil pouches or thelike) that can be separated by the user (e.g., by tearing on score linesbetween the two containers).

In still another embodiment, the invention provides a kit comprising inseparate vessels, a) a sirtuin modulator of this invention; and b)another therapeutic agent such as those described elsewhere in thespecification.

The practice of the present methods will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2^(nd) Ed., ed. by Sambrook, Fritsch andManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention inany way.

Example 1 Preparation ofN-(2-chloro-5-methylpyridin-3-yl)-2-nitrobenzamide

To a solution of 5-amino-6-chloro-3-picoline (9.54 g, 66.9 mmol) inpyridine (200 mL) was added 2-nitrobenzoyl chloride (13.65 g, 73.6 mmol)dropwise at 0° C. The resulting mixture was stirred at room temperature(18 h). The dark mixture was then diluted with water (1500 mL) and sat.sodium bicarbonate solution was added until the pH reached 8. Theprecipitate was collected by filtration, rinsed with water (30 mL×3) anddried to afford N-(2-chloro-5-methylpyridin-3-yl)-2-nitrobenzamide as apale solid (17.70 g, yield: 91%).

Preparation of 6-methyl-2-(2-nitrophenyl)thiazolo[5,4-b]pyridine

A mixture of N-(2-chloro-5-methylpyridin-3-yl)-2-nitrobenzamide (5.0 g,17.1 mmol) and P₂S₅ (7.6 g, 34.2 mmol) in pyridine (50 mL) and p-xylene(200 mL) was stirred at 140° C. (20 h). The solvent was removed invacuo. The residue was purified by recrystallization from EtOH to afford6-methyl-2-(2-nitrophenyl)thiazolo[5,4-b]pyridine as a yellow solid (3.5g, yield: 75%).

Preparation of 6-(bromomethyl)-2-(2-nitrophenyl)thiazolo[5,4-b]pyridine

6-Methyl-2-(2-nitrophenyl)thiazolo[5,4-b]pyridine (2.9 g, 10.7 mmol),N-bromosuccinimide (NBS, 1.91 g, 10.7 mmol), CCl₄ (200 mL) and benzoylperoxide (0.021 g) were combined in a three-neck flask (500 mL) underargon. The resulting yellow mixture was stirred at reflux (2 h).Additional NBS (1.91 g) and benzoyl peroxide (0.021 g) were added. Afteran additional 2 h at reflux, NBS (0.95 g) and benzoyl peroxide (0.021 g)were again added and the mixture was refluxed for 3 h. The mixture wasallowed to cool to room temperature and concentrated in vacuo to affordcrude 6-(bromomethyl)-2-(2-nitrophenyl)thiazolo[5,4-b]pyridine (4.0 g),which was taken directly to the next step.

Preparation of tert-butyl4-((2-(2-nitrophenyl)thiazolo[5,4-b]pyridin-6-yl)methyl)piperazine-1-carboxylate

A solution of crude6-(bromomethyl)-2-(2-nitrophenyl)thiazolo[5,4-b]pyridine (4.0 g),Boc-piperazine (1.99 g, 10.7 mmol), triethylamine (Et₃N) (1.5 mL, 10.7mmol) and acetonitrile (100 mL) was stirred at 50° C. (4 h) and then atroom temperature (60 h). TLC demonstrated that the reaction wascomplete. The mixture was concentrated in vacuo and purified by silicagel chromatography (petroleum ether:ethyl acetate:Et₃N=100:10:1) toafford tert-butyl4-((2-(2-nitrophenyl)thiazolo[5,4-b]pyridin-6-yl)methyl)piperazine-1-carboxylateas a yellow solid (3.6 g, yield: 74% over 2 steps).

Preparation of tert-butyl4-((2-(2-aminophenyl)thiazolo[5,4-b]pyridin-6-yl)methyl)piperazine-1-carboxylate

A mixture of tert-butyl4-((2-(2-nitrophenyl)thiazolo[5,4-b]pyridin-6-yl)methyl)piperazine-1-carboxylate(2.13 g, 4.7 mmol), NH₄Cl (2.00 g, 37 mmol), iron powder (1.31 g, 23.5mmol), H₂O (40 mL) and methanol (160 mL) was refluxed for 3 h under N₂.The reaction mixture was filtered, and the filtrate was concentrated invacuo and purified by silica gel chromatography (petroleum ether:ethylacetate:Et₃N=800:200:1) to afford tert-butyl4-((2-(2-aminophenyl)thiazolo[5,4-b]pyridin-6-yl)methyl)piperazine-1-carboxylateas a yellow solid (1.63 g, yield: 81%).

The aniline intermediates for compounds 1-6, 11-43, 45, 47-61, 65,67-68, 72, 74-75, 77-88, 90, 92-122, 127-130 were prepared in ananalogous manner as that outlined above, using appropriate aminereagents.

Preparation ofN-(2-(6-(piperazin-1-ylmethyl)thiazolo[5,4-b]pyridin-2-yl)phenyl)-3-(1H-tetrazol-5-yl)benzamide

tert-Butyl4-((2-(2-aminophenyl)thiazolo[5,4-b]pyridin-6-yl)methyl)piperazine-1-carboxylate(85 mg, 0.2 mmol) was taken up in 3 mL of DMF along with3-(1H-tetrazol-5-yl)benzoic acid (38 mg, 0.2 mmol), HATU (114 mg, 0.3mmol) and DIEA (35 mL, 0.4 mmol). The reaction mixture was stirred at50° C. (18 h). It was then diluted with EtOAc (15 mL) and washed withwater (3 mL×3). The organic layer was dried (Na₂SO₄) and concentrated invacuo to afford crude tert-butyl4-((2-(2-(3-(1H-tetrazol-5-yl)benzamido)phenyl)thiazolo[5,4-b]pyridin-6-yl)methyl)piperazine-1-carboxylate.This material was taken up in 5 mL of 25% TFA in CH₂Cl₂ and was allowedto stand at room temperature (18 h). The reaction mixture wasconcentrated in vacuo and the resulting residue was purified byreverse-phase HPLC to afford 12 mg ofN-(2-(6-(piperazin-1-ylmethyl)thiazolo[5,4-b]pyridin-2-yl)phenyl)-3-(1H-tetrazol-5-yl)benzamideas a light yellow solid. ¹H NMR (300 MHz, DMSO-d₆) δ 9.5 (br s, 2H),7.5-9.1 (m, 10H), 4.5 (br s, 1H), 3.5-4.0 (m, 10H). MS (ESI) calcd forC₂₅H₂₃N₉OS (m/z): 497.17, found: 498.2 (M+1)⁺.

All of the compounds discussed in this patent were prepared in ananalogous manner as that outlined above, using the appropriate acids.Those products which did not contain a BOC-protecting group did notproceed through the TFA deprotection step.

Example 2 Preparation of 1-methyl-4-phenyl-1H-pyrrole-2-carboxylic Acid

To a stirred solution ofmethyl-4-bromo-1-methyl-1H-pyrrole-2-carboxylate (2.5 g, 11.5 mmol) inDMF (35 mL) under argon were added sequentially Pd(PPh₃)₄ (0.66 g, 0.58mmol) and phenyl boronic acid (3.46 g, 28.4 mmol). The reaction mixturewas heated to 70° C., and Na₂CO₃ (11 g, 104 mmol) dissolved in water (30mL) was added. The reaction mixture was heated to 110° C. (14 h), cooledto room temperature, diluted with water (250 mL), and extracted withdiethyl ether (250 mL×3). The combined organic layers were dried andconcentrated in vacuo. The residue was purified by silica chromatography(eluted with petroleum ether:ethyl acetate=40:1) to affordmethyl-1-methyl-4-phenyl-1H-pyrrole-2-carboxylate as a white crystallinesolid (0.257 g). The ester (0.26 g, 1.2 mmol) was then combined withLiOH/H₂O (0.15 g, 3.6 mmol) in THF (5 mL), CH₃OH (5 mL) and H₂O (2.5 mL)and was stirred at 40° C. (4 h). Water (25 mL) was added and the mixturewas washed with DCM (15 mL). The pH of the aqueous phase was adjusted to5-7 using HCl (1N). The water was removed and the residue was dissolvedin MeOH (100 mL) and then filtered. The filtrate was concentrated invacuo to give 1-methyl-4-phenyl-1H-pyrrole-2-carboxylic acid as whitesolid. (0.419 g) ¹H-NMR (CDCl₃, 400 MHz): δ 4.01 (s, 3H), 7.16 (d, J=1.6Hz, 1H), 7.23 (t, J=7.2 Hz, 1H), 7.38 (m, 3H), 7.53 (dd, J=7.2, 8.4 Hz,2H)

Example 3 Preparation of 2-phenylisonicotinic Acid

2-Bromoisonicotinic acid (2.02 g, 10 mmol) was dissolved in DMF (80 mL)under argon. Pd(PPh₃)₄ (0.6 g, 0.52 mmol) was added, and the reactionmixture was stirred at room temperature for 15 min. Na₂CO₃ (aq. 2N, 40mL) was then added, followed by the addition of phenylboronic acid (1.67g, 13.7 mmol). The reaction mixture was heated at 95° C. (18 h), cooledto room temperature and filtered through a celite pad. Water (80 mL) wasadded, and the mixture was acidified with HCl (2 N) to pH=4. Theprecipitate was collected via filtration and rinsed with water (2×7 mL).The crude product was recrystallized from 2-methoxylethanol to give2-phenylisonicotinic acid as a grey solid (1.2 g). ¹HNMR (DMSO-d6, 400MHz): δ 7.51 (m, 3H), 7.78 (d, J=4.8 Hz, 1H), 8.13 (t, J=1.6 Hz, 2H),8.29 (s, 1H), 8.85 (d, J=4.8 Hz, 1H), 13.73 (bs, 1H).

Example 4 Biological Activity

A mass spectrometry based assay was used to identify modulators of SIRT1activity. The mass spectrometry based assay utilizes a peptide having 20amino acid residues as follows:Ac-EE-K(biotin)-GQSTSSHSK(Ac)NleSTEG-K(5TMR)-EE-NH2 (SEQ ID NO: 1)wherein K(Ac) is an acetylated lysine residue and Nle is a norleucine.The peptide is labeled with the fluorophore 5TMR (excitation 540nm/emission 580 nm) at the C-terminus. The sequence of the peptidesubstrate is based on p53 with several modifications. In addition, themethionine residue naturally present in the sequence was replaced withthe norleucine because the methionine may be susceptible to oxidationduring synthesis and purification.

The mass spectrometry assay is conducted as follows: 0.5 μM peptidesubstrate and 120 μM βNAD⁺ is incubated with 10 nM SIRT1 for 25 minutesat 25° C. in a reaction buffer (50 mM Tris-acetate pH 8, 137 mM NaCl,2.7 mM KCl, 1 mM MgCl₂, 5 mM DTT, 0.05% BSA). Test compounds may beadded to the reaction as described above. The SirT1 gene is cloned intoa T7-promoter containing vector and transformed into BL21(DE3). Afterthe 25 minute incubation with SIRT1, 10 μL of 10% formic acid is addedto stop the reaction. Reactions are sealed and frozen for later massspec analysis. Determination of the mass of the substrate peptide allowsfor precise determination of the degree of acetylation (i.e. startingmaterial) as compared to deacetylated peptide (product).

A control for inhibition of sirtuin activity is conducted by adding 1 μLof 500 mM nicotinamide as a negative control at the start of thereaction (e.g., permits determination of maximum sirtuin inhibition). Acontrol for activation of sirtuin activity is conducted using 10 nM ofsirtuin protein, with 1 μL of DMSO in place of compound, to determinethe amount of deacetylation of the substrate at a given timepoint withinthe linear range of the assay. This timepoint is the same as that usedfor test compounds and, within the linear range, the endpoint representsa change in velocity.

For the above assay, SIRT1 protein was expressed and purified asfollows. The SirT1 gene was cloned into a T7-promoter containing vectorand transformed into BL21(DE3). The protein was expressed by inductionwith 1 mM IPTG as an N-terminal His-tag fusion protein at 18° C.overnight and harvested at 30,000×g. Cells were lysed with lysozyme inlysis buffer (50 mM Tris-HCl, 2 mM Tris[2-carboxyethyl]phosphine (TCEP),10 μM ZnCl₂, 200 mM NaCl) and further treated with sonication for 10 minfor complete lysis. The protein was purified over a Ni-NTA column(Amersham) and fractions containing pure protein were pooled,concentrated and run over a sizing column (Sephadex S200 26/60 global).The peak containing soluble protein was collected and run on anIon-exchange column (MonoQ). Gradient elution (200 mM-500 mM NaCl)yielded pure protein. This protein was concentrated and dialyzed againstdialysis buffer (20 mM Tris-HCl, 2 mM TCEP) overnight. The protein wasaliquoted and frozen at −80° C. until further use.

Sirtuin modulating compounds that activated SIRT1 were identified usingthe assay described above and are shown below in Table 3. The EC_(1.5)values represent the concentration of test compounds that result in 150%activation of SIRT1. NT means that the compound was not tested using theindicated assay. NA means that the compound was not active in theindicated assay. The percent maximum fold activation of SIRT1 is alsoindicated in Table 3.

TABLE 3 COMPOUND EC_(1.5) % FOLD NO [M + H]+ STRUCTURE NM ACT. 1 507

233 241 2 498

NA 132 3 497

1620 246 4 508.1

504 321 5 508

665 281 6 508

746 274 7 579

65 581 8 507

340 471 9 507

416 392 10 507

492 316 11 520

78 578 12 521

249 550 13 491

584 252 14 480

937 381 15 535.1

627 531 16 535.2

674 405 17 535.3

683 360 18 521.2

620 457 19 479.2

722 198 20 492.2

687 224 21 521.2

162 906 22 480.2

2620 417 23 480.1

1950 241 24 534.2

94 299 25 509

469 203 26 510

455 192 27 510

913 185 28 480.2

3590 162 29 519.2

462 378 30 526

986 142 31 511

1000 168 32 530

815 162 33 564.2

91 533 34 565.2

281 492 35 565.2

554 234 36 506.1

NT NT 37 507.1

507 422 38 509

548 292 39 528

400 241 40 497

182 397 41 585.2

652 275 42 566.2

656 212 43 499

215 236 44 496

397 603 45 554.2

144 569 46 527

503 446 47 565.2

82 1032 48 525.1

395 222 49 525.1

319 192 50 526.1

533 207 51 526.1

1640 172 52 526.1

357 411 53 526.1

66 837 54 528.1

110 538 55 508.1

74 >500 56 565.2

458 389 57 583.2

41 >500 58 565.2

884 370 59 565.2

492 279 60 492.2

144 477 61 510.2

78 >500 62 497

592 231 63 482

859 303 64 481

260 >500 65 483

NA 100 66 483

819 336 67 477

495 322 68 482

593 295 69 481

687 >500 70 488

2590 298 71 496

1460 304 72 509

NA 100 73 508

2970 183 74 527

421 236 75 515

NT NT 76 498

2790 285 77 499

3920 204 78 542.1

565 297 79 528.1

1220 240 80 529.1

NA 100 81 556.2

NA 131 82 528.1

878 224 83 501.1

NA 100 84 501

4270 166 85 586.2

16700 182 86 565.2

NA 137 87 566.2

955 166 88 567.2

14 272 89 484

235 239 90 495.2

190 221 91 487.3

4410 333 92 441

2810 163 93 488.1

510 236 94 463

150 182 95 466.1

160 252 96 481

1060 162 97 481

3020 186 98 472

NA 139 99 496.1

110 244 100 484

1440 201 101 502

NA 133 102 516

NA 131 103 495

230 273 104 482

200 221 105 512

1590 292 106 482

69840 189 107 483

500 219 108 498

1060 178 109 481

270 >500 110 482

230 307 111 471

1610 427 112 472

2330 375 113 484

370 232 114 498

NA 146 115 495

620 >500 116 495

270 >500 117 510

480 324 118 516

NA 100 119 481

390 >500 120 481

820 >500 121 496

750 291 122 481

NT NT 123 470

1940 400 124 488

1060 314 125 505

760 437 126 489

NA 100 127 505

NA 143 128 489

1310 227 129 541

NA 100 130 540

990 161 131

106 500 132

215 500 133

90 500 134

69 500 135

759 274 136

448 461 137

214 448 138

88 497 139

230 457 140

41 500 141

127 487 142

394 378 143

2121 372 144

559 402 145

86 500 146

127 500 147

845 224 148

40 500 149

557 216 150

67 267 151

591 170 152

321 >500 153

829 226 154

428 277 155

141 364 156

674 443

EQUIVALENTS

The present invention provides among other things sirtuin-activatingcompounds and methods of use thereof. While specific embodiments of thesubject invention have been discussed, the above specification isillustrative and not restrictive. Many variations of the invention willbecome apparent to those skilled in the art upon review of thisspecification. The full scope of the invention should be determined byreference to the claims, along with their full scope of equivalents, andthe specification, along with such variations.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein, including those itemslisted below, are hereby incorporated by reference in their entirety asif each individual publication or patent was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

Also incorporated by reference in their entirety are any polynucleotideand polypeptide sequences which reference an accession numbercorrelating to an entry in a public database, such as those maintainedby The Institute for Genomic Research (TIGR) (www.tigr.org) and/or theNational Center for Biotechnology Information (NCBI)(www.ncbi.nlm.nih.gov).

Also incorporated by reference are the following: PCT Publications WO2005/002672; 2005/002555; and 2004/016726.

1. A compound represented by Structural Formula (II):

or a salt thereof, wherein: two of X¹ to X⁴ are selected from —CR*— and—N—; the other two of X¹ to X⁴ are —CR*—; R* is independently selectedat each occurrence from —H, lower alkyl or halogen; R¹ is a solubilizinggroup; and R² is selected from phenyl optionally substituted with one ormore substituents independently selected from —CN, —F, —Cl and —CF₃, andwhen each of X¹ to X⁴ is —CR*—, R² is additionally selected from a 5- to6-membered heterocycle containing an N heteroatom and, optionally, asecond heteroatom selected from N, O or S, wherein said heterocycle isoptionally substituted with methyl.
 2. The compound of claim 1, wherein:X¹ to X⁴ is —CR*—; and R² is selected from phenyl, fluorophenyl,difluorophenyl, chlorophenyl, difluorophenylmethylthiazolyl,pyrimidinyl, pyridyl and pyrazolyl.
 3. The compound of claim 2, whereinR² is selected from phenyl, fluorophenyl, difluorophenyl, chlorophenyl,2-methylthiazol-4-yl, pyridyl and pyrazol-1-yl.
 4. The compound of claim3, wherein R² is phenyl or pyridyl.
 5. The compound of claim 1, whereinone of X¹ to X⁴ are —N—.
 6. The compound of claim 1, wherein two of X¹to X⁴ are —N—.
 7. The compound of claim 5, wherein X¹ is N.
 8. Thecompound of claim 6, wherein X¹ and X² are —N—.
 9. The compound of claim6, wherein X¹ and X⁴ are —N—.
 10. The compound of claim 1, wherein: R¹is —CH₂—R³; and R³ is a nitrogen-containing heterocycle optionallysubstituted with one or more substituents selected from C₁-C₄ alkyl,amino, halogen, methoxy and methoxy-C₁-C₄ alkyl.
 11. The compound ofclaim 10, wherein R² is phenyl, pyridyl or 3-fluorophenyl.
 12. Thecompound of claim 11, wherein X² and X³ are —CH— and X¹ and X⁴ areindependently selected from —CR*— or —N—.
 13. The compound of claim 10,wherein: R¹ is —CH₂—R³; and R³ is selected from piperazin-1-yl,4-(methoxyethyl-piperazin-1-yl, 3,5-dimethylpiperazin-1-yl,morpholin-4-yl, piperidin-1-yl, 4-aminopiperidin-1-yl, pyrrolidin-1-yl,3-fluoropyrrolidin-1-yl, —NH-(pyrrolidin-3-yl), and1,4-diaza-bicyclo[2.2.1]heptan-1-yl.
 14. The compound of claim 13,wherein R³ is selected from 4-(methoxyethyl)-piperazin-1-yl,morpholin-4-yl, piperidin-1-yl and 4-aminopiperidin-1-yl.
 15. Thecompound of claim 14, wherein R² is phenyl, 3-fluorophenyl or pyridyl.16. The compound of claim 15, wherein X² and X³ are —CH— and X¹ and X⁴are independently selected from —CR*— or —N—.
 17. The compound of claim1, wherein R* is H.
 18. The compound of claim 1, represented by formula(IV):

or a salt thereof, wherein: one X variable is selected from —CH— and—N—; the other two X variables are —CH—; R¹ is a solubilizing group; andR² is selected from phenyl and fluorophenyl, and, when each X variableis —CH—, R² is additionally selected from a 5- to 6-membered heterocyclecontaining an N heteroatom and, optionally, a second heteroatom selectedfrom N, O or S, wherein said heterocycle is optionally substituted withmethyl.
 19. A compound represented by Structural Formula (V):

or a salt thereof, wherein: ring A is selected from:

R¹ is a solubilizing group; and R^(#) is a —H or —O—CH₃.
 20. A compoundrepresented by Structural Formula (VI):

or a salt thereof, wherein: ring B is selected from:

R¹ is a solubilizing group.
 21. A pyrogen-free composition comprising acompound of any one of claims 1, 19 or 20, or a pharmaceuticallyacceptable salt thereof and a carrier.
 22. A pharmaceutical compositioncomprising a compound of any of one claims 1, 19 or 20 and apharmaceutically acceptable carrier.
 23. The pharmaceutical compositionof claim 22, further comprising an additional active agent.
 24. A methodfor treating a subject suffering from or susceptible to insulinresistance, a metabolic syndrome, diabetes, or complications thereof, orfor increasing insulin sensitivity in a subject, comprisingadministering to the subject in need thereof a composition of claim 22.25. The method of claim 24, wherein said compound increases at least oneof the level or activity of a sirtuin protein.
 26. The method of claim25, wherein the compound increases deacetylase activity of the sirtuinprotein.
 27. The method of claim 25, wherein the sirtuin protein is amammalian protein.
 28. The method of claim 25, wherein the sirtuinprotein is human SIRT1.
 29. The method of claim 25, wherein the compounddoes not substantially have one or more of the following activities:inhibition of PI3-kinase, inhibition of aldoreductase, inhibition oftyrosine kinase, transactivation of EGFR tyrosine kinase, coronarydilation, or spasmolytic activity, at concentrations of the compoundthat are effective for increasing the deacetylation activity of a SIRT1and/or SIRT3 protein.